NO20150196L - Method for detecting and / or identifying adeno-associated virus (AAV) sequences and isolating new sequences thus identified. - Google Patents

Abstract

A method for detecting and isolating AAV sequences in a sample of DNA obtained from tissues or cells is described. Further described are AAV sequences identified by this method as well as vectors constructed using these sequences.

Description

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is a small, non-enveloped, icosahedral virus with single-stranded linear DNA genomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus Dependovirus because the virus was discovered as a contaminant in purified adenovirus stocks. The AAV life cycle includes a latent phase in which AAV genomes, after infection, become site-specifically integrated into host chromosomes, and an infectious phase in which, after either adenovirus or herpes simplex virus infection, the integrated genomes are then rescued, replicated and packaged into infectious vims . The properties of non-pathogenicity, broad spectrum of infectivity including non-dividing cells, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.

Recent studies suggest that AAV vectors may be the vehicle of choice for gene therapy. To date, 6 different serotypes of AAVs have been isolated from human or non-human primates (NHP) and these are well characterized. Among these, human serotype 2 is the first AAV to be developed as a gene transfer vector; it has subsequently been extensively used for efficient gene transfer experiments in various target tissues and animal models. Clinical trials of experimental application of AAV2-based vectors against certain human disease models are in progress and include diseases such as cystic fibrosis and haemophilia B.

The desire is to provide AAV-based constructs for gene delivery.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a new method for detecting and identifying AAV sequences from cellular DNAs of various human and non-human primate (NHP) tissues using bioinformatics analyses, PCR-based gene amplification and cloning technology, based on the nature of latency and integration of AAVs in the absence of helper virus ruscoinfection.

In a further aspect, the present invention provides methods for isolating newly detected AAV sequences using the above-described method according to the invention. The invention further includes methods for generating vectors based on these new AAV serotypes for serology and gene transfer studies based only on the availability of capsid gene sequences and the structure of rep/cap gene splices.

In yet another aspect, the present invention provides a new method for carrying out studies in connection with serology, epidemiology, biodistribution and mode of transmission using reagents according to the invention which include generic sets of primers/probes and quantitative real-time PCR.

In yet another aspect, the invention provides a method for isolating complete and infectious genomes of recent AAV serotypes from cellular DNA and diverse origins using RACE and other molecular techniques.

In yet another aspect, the invention provides a method for arriving at new serotypes of AAV genomes from human and NHP cell lines using adenovirus helpers of different origins.

In yet another aspect, the invention provides new AAV serotypes, vectors containing these and methods for using them.

These and other aspects according to the invention will appear from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A to 1AAAR provide an arrangement of the nucleic acid sequence encoding at least the cap proteins for the AAV serotypes. The full-length sequences including the ITRs, the rep region and the capsid region are provided for new AAV serotypes 7 [SEQ ID No. 1], and for previously published AAV1 [SEQ IN No. 6], AAV2 [SEQ ID NO: 7]; and AAV3 [SEQ ID NO: 8]. New AAV serotypes AAV8 [SEQ ID no. 4] and AAV9 [SEQ ID no. 5] are the subject of simultaneously submitted applications. The other novel clones of the invention provided in this facility include: 42-2 [SEQ ID No. 9], 42-8 [SEQ ID

No. 27], 42-15 [SEQ ID No. 28], 42-5b [SEQ ID No. 29], 42-lb [SEQ ID No. 30]; 42-13 [SEQ ID No. 31], 42-3a [SEQ ID No. 32], 42-4 [SEQ ID No. 33], 42-5a [SEQ ID No. 34], 42-10 [SEQ ID No. 35], 42-3b [SEQ ID No. 36], 42-11 [SEQ ID No. 37], 42-6b [SEQ ID No. 38], 43-1 [SEQ ID No. 39], 43 -5 [SEQ ID No. 40], 43-12 [SEQ ID No. 41], 43-20 [SEQ ID No. 42], 43-21 [SEQ ID No. 43], 43-23 [SEQ ID No. .44], 43-25 [SEQ ID No. 45], 44.1 [SEQ ID No. 47], 44.5 [SEQ ID No. 47], 223.10 [SEQ ID No. 48], 223.2 [SEQ ID No. 49] , 223.4 [SEQ ID No. 50], 223.5 [SEQ ID No. 51], 223.6 [SEQ ID No. 52], 223.7 [SEQ ID No. 53], A3.4 [SEQ ID No. 54], A3. 5 [SEQ ID No. 55], A3.7 [SEQ ID No. 56],

A3.3 [SEQ ID No. 57], 42.12 [SEQ ID No. 58], 44.2 [SEQ ID No. 59]. The nucleotide sequences of the signature regions of AAV10 [SEQ ID No. 117], AAV11 [SEQ ID No. 118] and AAV12 [SEQ ID No. 119] are provided in this figure. Critical landmarks in the structure of the AAV genomes are shown. Gaps are indicated by points. The 3' ITR of AAV1 [SEQ ID No. 6] is shown in the same configuration as in the published sequences. TRS represents the thermal resolution seat. Note that AAV7 is the only AAV reported to use GTG as the initiation codon for VP3.

Figures 2A to 2F are alignments of the amino acid sequences of the proteins of the vpl capsid proteins of previously published AAV serotypes 1 [SEQ ID No. 64], AAV2 [SEQ ID No. 70], AAV3 [SEQ ID No. 71], AAV4 [SEQ ID No. 63], AAV5 [SEQ ID No. 114] and

AAV6 [SEQ ID No. 65] and novel AAV sequences according to the invention, including: Cl [SEQ ID No. 60], C2 [SEQ ID No. 61], C5 [SEQ ID No. 62], A3-3 [SEQ ID No. 66], A3-7 [SEQ ID No. 67], A3-4 [SEQ ID No. 68], A3-5 [SEQ ID No. 69], 3.3b [SEQ ID No. 62], 223.4 [ SEQ ID No. 73], 223-5 [SEQ ID No. 74], 223-10 [SEQ ID No. 75], 223-2 [SEQ ID No. 76], 223-7 [SEQ ID No. 77] , 223-6 [SEQ ID No. 78], 44-1 [SEQ ID No. 79], 44-5 [SEQ ID No. 80], 44-2 [SEQ ID No. 81], 42-15 [SEQ ID No. 84], 42-8 [SEQ ID No. 85], 42-13 [SEQ ID No. 86], 42-3A [SEQ ID No. 87], 42-4 [SEQ ID No. 88], 42-5A [SEQ ID No. 89], 42-1B [SEQ ID No. 90], 42-5B [SEQ ID No. 91], 43-1 [SEQ ID No. 92], 43-12 [SEQ ID No. 93], 43-5 [SEQ ID No. 94], 43-21 [SEQ ID No. 96], 43-25 [SEQ ID No. 97], 43-20 [SEQ ID No. 99], 24.1 [SEQ ID No. 101], 42.2 [SEQ ID No. 102], 7.2 [SEQ ID No. 103], 27.3 [SEQ ID No. 104], 16.3 [SEQ ID No. 105], 42.10 [SEQ ID No. 106], 42-3B [SEQ ID No. 107], 42-11 [SEQ ID No. 108], Fl [SEQ ID No. 109], 5F [SEQ ID No. 110], F3 [S EQ ID No. 111], 42-6B [SEQ ID No. 112], 42-12 [SEQ ID No. 113].

New serotypes AAV8 [SEQ ID No. 95] and AAV9 [SEQ ID No. 100] are the subject of concurrently filed applications.

Figures 3A to 3C provide the amino acid sequence of the AAV7 rep proteins [SEQ ID No. 3].

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the present inventors have found a method that takes advantage of the ability of adeno-associated virus (AAV) to penetrate the nucleus and, in the absence of a helper virus co-infection, to integrate into cellular DNA and establish a latent infection. This method uses a polymerase chain reaction (PCR)-based strategy for the detection, identification and/or isolation of sequences of AAVs from DNAs from tissues of human and non-human primate origin as well as from other sources. Preferably, this method is also suitable for detecting, identifying and/or isolating other integrated, viral and non-viral sequences, as described below.

The invention further provides nucleic acid sequences which have been identified according to the method of the invention. One such adeno-associated virus is of a new serotype, here called serotype 7 (AAV7). Other novel adeno-associated virus serotypes provided herein also include AAV 10, AAV11 and AAV12. Further other AAV serotypes which have been identified according to the methods of the invention are provided in the present description, reference is made to the figures and the sequence listing which are part of the description.

Further fragments of these AAV sequences are provided. Among particularly desired AAV fragments are the cap proteins including vi, vp2, vp3, the hypervariable regions, the rep proteins including rep 78, rep 68, rep 52 and rep 40, and the sequences encoding these proteins. Each of these fragments can easily be used in different vector systems and host cells. Such fragments can be used alone, in combination with other AAV sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. In a particularly desirable embodiment, a vector contains the AAV cap and/or rep sequences according to the invention.

As described herein, alignment is performed using any of a number of commonly or commercially available "Multiple Sequence Alignment Programs" such as "Clustal W", available via Web Servers on the internet. Alternatively, vector NTI utilities are also used. There are also a number of algorithms well known in the art that can be used to measure nucleotide sequence identity including those contained in the programs described above. As another example, polynucleotide sequences can be compared using Fasta, a program in GCG Version 6.1. Fasta provides means and percent sequence identity for the regions of best overlap between the question and answer sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (a word size of 6 and the NOPAM factor for the evaluation matrix) as provided in GCG Version 6.1, to which reference is made for details. Similar programs are available for amino acid sequences, for example the "Clustal X" program. In general, any of these programs can be used as default settings, although the person skilled in the art can change these as needed. Alternatively, the person skilled in the field can use other algorithms or machine programs that provide at least the level of identity or facility provided by said algorithms and programs. The term "substantial homology" or "substantial similarity" indicates that, with reference to a nucleic acid or fragment thereof, and upon optimal alignment with suitable nucleotide insertions or deletions with other nucleic acids (or its complementary strand), there is nucleotide sequence identity of at least about 95 to 99% of the aligned sequences. Preferably, the homology is over the full-length sequence, or an open reading frame thereof, or another suitable fragment that is at least 15 nucleotides in length. Examples of suitable fragments are described here.

The term "substantial homology" or "substantial similarity" indicates that, with reference to amino acids or fragments thereof and when optimally aligned with suitable amino acid insertions or deletions with other amino acids, there is amino acid sequence identity of at least about 95 to 99% of the aligned sequences. Preferably, the homology is over full-length sequences or a protein thereof, for example a cap protein, a rep protein or a fragment thereof of at least 8 amino acids or preferably at least 15 amino acids in length. Examples of suitable fragments are described here.

The term "highly preserved" or similar means at least 80% identity, preferably at least 90% identity and most preferably more than 97% identity. Identity is easily determined by the person skilled in the art by using algorithms and computer programs known to the person skilled in the art.

The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to residues in the two sequences that are the same when aligned for maximum agreement. The length of the sequence identity comparison can be over the entire length of the genome, the entire length of a gene coding sequence, or a fragment of at least 500 to 5000 nucleotides, if desired. However, identity among smaller fragments, for example at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, or at least about 36 or more nucleotides, may also be desirable. Likewise, "percent sequence identity" can be easily determined for amino acid sequences over the entire length of a protein, or a fragment thereof. Conveniently, a fragment is at least 8 amino acids in length and can be up to 700 amino acids. Examples of suitable fragments are described here.

The AAV sequences and fragments thereof are useful in the production of rAAV and are also useful as antisense delivery vectors, gene therapy vectors or vaccine vectors. The invention further provides nucleic acid molecules, gene delivery vectors and host cells containing the AAV sequences according to the invention.

As described here, the vectors according to the invention containing the AAV capsid proteins according to the invention are particularly well suited for use in applications where the neutralizing antibodies reduce the effectiveness of other AAV serotype-based vectors, as well as other viral vectors. The rAAV vectors according to the invention are particularly advantageous for rAAV re-administration and repeat gene therapy.

These and other embodiments and advantages according to the invention are described in greater detail below.

As used in the description and claims, the terms "comprehensive" and "including" and their variations are inclusive of other components, elements, integers, steps and the like. Conversely, the term "consisting of" and variants thereof are exclusive of other components, elements, integers, steps and the like.

I. Methods according to the invention

A. Detection of sequences via molecular cloning

In one aspect, the invention provides a method for detecting and/or identifying large nucleic acid sequences in a sample. This test is particularly suitable for the detection of viral sequences that are integrated into a cell's chromosome, for example adeno-associated viruses (AAV) and retroviruses, among others. The description refers to AAV, which is exemplified here. Based on this information, however, the person skilled in the art will easily be able to carry out the methods according to the invention on retro vims [for example feline leukemia vi-ms (FeLV), HTLVI and HTLVUI], and lentivirinae [for example human immunodeficient vims (FflV), simian immunodeficient vims (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, and spumavirinal)], among others. Furthermore, the method according to the invention can also be used for the detection of other viral and non-viral sequences, regardless of whether they are integrated or not integrated into the genome of the host cell.

As used herein, a sample is any source containing nucleic acids, such as tissue, tissue culture, cells, cell culture, and biological fluids including, without limitation, urine and blood. These nucleic acid sequences can be DNA or RNA from plasmids, natural DNA or RNA from any source including bacteria, yeast, fungi and higher organisms such as plants or animals. DNA or RNA is extracted from samples by a number of techniques well known to those skilled in the art and such as those described by Sambrook in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory). The origin of the sample and the method by which the nucleic acids are obtained for carrying out the method according to the invention is not a limitation for this. Optionally, the methods of the invention can be carried out directly on the source of DNA, or on obtained nucleic acids (which have, for example, been extracted) from a source.

The method according to the invention involves subjecting a sample containing DNA to amplification via polymerase chain reaction (PCR) using a first set of primers specific for a first region of double-stranded nucleic acid sequences in order to thereby obtain amplified sequences. As used herein, each of the "regions" is determined in advance based on the alignment of the nucleic acid sequence of at least two serotypes (for example, AAV) or strains (for example, lentiviruses) and where each of the regions consists of sequences with a 5' end that is highly conserved , a middle tree which is preferably but not necessarily variable, and a 3' end which is highly conserved in that each of these is conserved or variable relative to the sequences of the at least two aligned AAV serotypes. Preferably, the 5- and/or 3-end is highly conserved in relation to at least 9 and most preferably at least 18 base pairs (bp). However, one or both of the sequences at the 5- or 3-end may be conserved over more than 18 bp, more than 25 bp, more than 30 bp, or more than 50 bp at the 5-end. With regard to the variable region, there is no requirement for conserved sequences, these sequences may be relatively conserved or may have less than 90, 80 or 70% identity among the aligned serotypes or strains.

Each of the regions can span over 100 bp to around 10 kilobase pairs in length. However, it is particularly desirable that one of the regions be a "signature region", that is, a region sufficiently unique to positively identify the amplified sequence as one from the target source. In one embodiment, for example, the first region has a length of around 250 bp and is sufficiently unique among known AAV sequences that it positively identifies the amplified region as being of AAV origin. Furthermore, the variable sequences within the area are sufficiently unique that they can be used to identify the serotype from which the amplified sequence originates. Once amplified (and thereby detected), the sequences can be identified by performing conventional restriction digestion and comparison with the restriction patterns for this region in any of AAV1, AAV2, AAV3, AAV4, AAV5, or AAV6, or the to AAV7, AAV10, AAV11, AAV12 or any other new serotype identified according to the invention, which is determined in advance and provided by the invention.

Given the guidelines provided herein, one skilled in the art can easily identify such regions among other integrated features to allow easy detection and identification of these sequences. Then, an optimal set of generic primers located at the highly conserved ends can be constructed and tested for efficient amplification of the selected regions from the samples. This aspect of the invention can be easily adapted to a diagnostic kit for detecting the presence of the target sequence (for example AAV) and for identifying the AAV serotype, by bmk of standards that include the restriction patterns for the AAV serotypes described herein or that are isolated by bmk of those described herein techniques. For example, rapid identification or molecular serotyping of PCR products can be carried out by digesting the PCR products and comparing restriction patterns.

Thus, in one embodiment, the "signature region" for AAV spans mnt bp 2800 to mnt 3200 of AAV1 [SEQ ID No. 6] and corresponding base pairs in AAV2, AAV3, AAV4, AAV5 and AAV6. More desirable is the region of approximately 250 bp, located within bp 2886 to mndt 3143 bp of AAV1 [SEQ ID No. 6] and corresponding base pairs in AAV2 [SEQ ID No. 7], AAV3 [SEQ ID No. 8] and other AAV serotypes , reference is made to figure 1. In order to allow rapid detection of AAV in the sample, primers are used that specifically amplify this signature region. However, the invention is not limited to the exact sequences identified here for the AAV signature region as the person skilled in the art can easily change the region to include a shorter fragment or larger fragments of this signature region.

The PCR primers are generated by bmk by techniques well known to those skilled in the art. Each of the PCR primer sets consists of a 5' primer and a 3' primer, see for example Sambrook et al supra. The term "primer" refers to a nucleotide which acts as an initiation point for synthesis when placed under conditions in which synthesis of a primer extension product complementary to the nucleic acid strand is induced. The primer is preferably single-stranded. If, however, a double-stranded primer is used, this is treated to separate the strands before they are used to make the extension products. The primers can be between 15 and 25 or more nucleotides and are preferably at least 18 nucleotides. For some applications, however, shorter nucleotides are used, for example 7 to 15 nucleotides.

The primers are chosen to be sufficiently complementary to the different strands of each specific sequence to be amplified to hybridize with their respective strands. The primer sequence therefore does not need to reflect the exact sequence of the region being amplified. For example, a non-complementary nucleotide fragment can be attached to the 5-end of the primer while the rest of the primer sequence is fully complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer provided that the primer sequence has sufficient complementarity with the sequence in the strand to be amplified to hybridize with it and form a template for synthesis of the extension product of the second primer.

The PCR primers for the signature region according to the invention are based on the highly conserved sequences of two or more aligned sequences (for example two or more AAV serotypes). The primers may match with less than exact identity among two or more aligned AAV serotypes at the 5' end or in the middle. However, sequence at the 3-end of the primers corresponds to a region of two or more aligned AAV serotypes in which there is exact identity over at least five and preferably over at least nine base pairs, most preferably over at least 18 base pairs at the 3-end of the primers. Thus, the 3-end of the primers consists of sequences with 100% identity to the aligned sequences over at least five nucleotides. However, one, two or more degenerate nucleotides can optionally be used at the 3-end of the primer.

For example, the primer set for the signature region of AAV was constructed based on a unique region within the AAV capsid as follows. The 5' primer end was based on nt 2867-2891 of AAV2 [SEQ ID No. 7], 5 -GGTAATTCCTCCGGAAATTGGCATT3'. See Figure 1.3' the primer end was constructed based on nt 3096-3122 of AAV2 [SEQ ID No. 7], 5-GACTCATCAACAACAACTGGGGATTC-3'. However, one skilled in the art can easily construct the primer set based on the corresponding regions of AAV1, AAV3, AAV4, AAV5, AAV6, or based on the formation provided therein, AAV7, AAAV10, AAV11, AAV12, or another new AAV according to the invention. In addition, additional other primer sets can be readily constructed to amplify this signature region using techniques well known to those skilled in the art.

B. Isolation of target sequences

As described herein, the invention provides a first primer set that specifically amplifies the signature region of the target sequence, for example an AAV serotype, to allow detection of the target. In a situation where additional sequences are desired, if for example a new AAV serotype is identified, the signature range can be extended. Thus, the invention can further use one or more additional primer sets.

Conveniently, these primer sets are designed to include either the 5' or 3' primer of the first primer set and a second primer unique to the primer set such that the primer set amplifies a region 5' or 3' to the signature region that anneals to either the 5' end or the 3 -end of the signature area. For example, a first primer set consists of a 5' primer, P1 and a 3' primer P2 to amplify the signature region. To extend the signature region at its 3 end, a second primer set consists of primer P1 and a 3' primer P4 which amplify the signature region and neighboring sequences downstream of the signature region. To extend the signature region at its 5 end, a third primer set consists of a 5' primer, P5, and primer 2 so that the signature region and neighboring sequences upstream of the signature region are amplified. These extension steps are repeated (or carried out simultaneously) as needed or desired. Then, the products resulting from these amplification steps are fused using conventional steps to yield an isolated sequence of the desired length.

The second and third primer sets are constructed as with the primer set for the signature region to amplify a region of highly conserved sequence among the aligned sequences.

References herein to the term "second" or "third" primer sets are for discussion only and without regard to the order in which these primers are added to the reaction mixture, or used for amplification. The region amplified by the second primer set is chosen so that upon amplification it anneals at its 5' end to the 3' end of the signature region. In a similar way, the region amplified by the third primer set is chosen so that upon amplification it anneals at its 3-end to the 5-end of the signature region. Additional primer sets can be designed so that the regions they amplify anneal to either the 5' end or the 3' end of the extension products formed by the second or third primer set, or by subsequent primer sets.

Where, for example, AAV is the target sequence, a first set of primers (P1 and P2) are used to amplify the signature region from the sample. In a desired embodiment, this signature region is located inside the AAV capsid. A second set of primers (P1 and P4) are used to extend the 3-end of the signature region to a location in the AAV sequence that is just in front of the AAV 3'ITR, i.e. which gives an extension product containing the entire 3-end of the AAV capsid when uses the signature area as an anchor. In one embodiment, the P4 primer corresponds to nt 4435 to 4462 of AAV2 [SEQ ID No. 7], and corresponding sequences in the other AAV serotypes. This results in the amplification of a region of around 1.6 kb containing the 0.25 kb signature region. A third set of primers (P3 and P2) is used to extend the 5-end of the signature region to a location in the AAV sequences that is at the 3-end of the rep genes, i.e. which gives an extension product containing the entire 5-end of the AAV capsid when the signature area is used as an anchor. In one embodiment, the P3 primer corresponds to nt 1384 to 1409 of AAV2 [SEQ ID No. 7] and corresponding sequences in other AAV serotypes. This results in amplification of a region of about 1.7 kb containing the 0.25 kb signature region. Optionally, a fourth set of primers is used to further extend the extension product containing the entire 5-end of the AAV capsid to also include the rep sequences. In one embodiment, P5 corresponds to nt 108 to 133 of AAV2 [SEQ ID no. 7] and corresponding sequences in the other AAV serotypes and is used in conjunction with the P2 primer.

After completion of the desired number of extension steps, the different extension products are fused using the signature region as an anchor or marker to construct an intact sequence. In the example given here, AAV sequences are obtained which as a minimum contain an intact AAV cap gene. Larger sequences can be achieved, depending on the number of relative steps that are carried out.

Appropriately, the extension products are assembled into an intact AAV sequence using methods well known to those skilled in the art. For example, the extension products can be digested with DraUI that cleaves at the DraUI site located in the signature region, to give restriction fragments that are re-ligated to give products that (at a minimum) contain an intact AAV cap gene. However, other suitable techniques for assembling the extension products into an intact sequence may be used. Reference is made here generally to Sambrook et al. supra.

As an alternative to the multiple extension steps described above, another embodiment of the invention provides direct amplification of a 3.1 kb fragment allowing isolation of full-length sequences. To directly amplify a 3.1 kb full-length cap fragment from NHP tissue and blood DNAs, two other highly conserved regions were identified in AAV genomes for use in PCR amplification of large fragments. A primer within a conserved region located in the middle of the rep gene is used (AVlns: 5' GCTGCGTCAACTGGACCAATGAGAAC 3', nt of SEQ ID no. 6) in combination with the 3' primer located in another conserved region downstream of the cap gene (AV2cas: 5' CGCAGAGACCAAAGTTCAACTGAAACGA 3', SEQ ID No. 7) for amplification of AAV sequences including the relevant full-length AAV cap. After amplification, the products are typically cloned and sequence analysis performed with an accuracy of >99.9%. By using this method, the present inventors have isolated at least 50 capsid clones which have then been characterized. Among these, 37 clones were derived from Rhesus macaque tissue (rh. 1 - rh.37), 6 clones from cynomologous macaques (cy. 1 - cy.6), 2 clones from baboons (bb.l and bb.2) and 5 clones from chimpanzees (ch.l - ch.5). These clones are identified elsewhere in the specification along with the animal species from which they were identified and the tissues in the animal where these new sequences were located.

C. Alternative method for isolation of new AAV

In another aspect, the invention provides an alternative method for isolating new AAV from a cell. This method involves infecting the cell with a vector that provides helper functions to the AAV in question; isolation of infectious clones containing AAV; sequencing the isolated AAV; and comparing the sequence of the isolated AAV with known AAV serotypes whereby differences in the sequences of the isolated AAV and known AAV serotypes indicate the presence of a new AAV.

In one embodiment, the vector that provides the helper function provides essential adenovirus functions including, for example, E1a, Elb, E2a, E40RF6.1 In one embodiment, these helper functions are provided by an adenovirus. The adenovirus may be a wild-type adenovirus and may be of human or non-human origin, preferably of non-human primate (NHP) origin. The DNA sequences of a number of adenovirus types are available from Genbank including type Ad5 [Genbank Accession no. M73260]. The adenovirus sequences can be obtained from any known adenovirus serotype such as serotypes 2, 3, 4, 7, 12 and 40, and further includes any of the human types so far identified, see for example Horwitz supra. Corresponding adenoviruses which are known to infect non-human animals, for example chimpanzees, can also be used in the vector constructs according to the invention, see for example US 6 083 716.1 addition to wild-type adenoviruses can recombinant vims or non-viral vectors (for example plasmids , episomes and so on) that carry the necessary helper functions are used. Such recombinant vims are well known in the art and can be prepared according to published techniques, see for example US 5,871,982 and 6,251,677 which describe a hybrid Ad/AAV vims. The choice of adenovim's type is not considered limiting for the invention. A number of adenovims strains are available from the American Type Culture Collection, Manassas, Virginia or available upon request from a number of commercial and institutional sources. Furthermore, the sequences for many such strains are available from a number of databases including, for example, PubMed and GenBank.

In another alternative, infectious AAV can be isolated by bmk by genome walking technology (Siebert et al., 1995, "Nucleic Acid Research", 23:1087-1088, Friezner-Degen et al., 1986, "J. Biol. Chem." 261: 6972-6985, BD Biosciences Clontech, Palo, Alto, CA). Genome walking is particularly well suited for the identification and isolation of sequences alongside the new sequences identified according to the method of the invention. For example, this technique may be useful for isolating inverted terminal repeats (ITRs) of the new AAV serotype, based on the new AAV capsid and/or rep sequences identified using the methods of the invention. This technique is also usable for isolating sequences next to other AAV and non-AAV sequences that have been identified and isolated according to the invention, see examples 3 and 4.

The methods according to the invention can easily be used for a number of epidemiological studies, studies of biodistribution, monitoring of gene therapy via AAV vectors and vectors derived from other integrated vims. Thus, the methods are well suited for use in prepackaged kits for bmk by doctors, researchers and epidemiologists.

n. Diagnostic kit

In another aspect, the invention provides a diagnostic kit for detecting the presence of a known or unknown adeno-associated virus (AAV) in a sample. Such a kit may contain a first set of 5- and 3' PCR primers specific for the signature region of the AAV nucleic acid sequence. Alternatively or additionally, such a kit may contain a first set of 5' and 3' PCR primers specific for the 3.1 kb fragment that includes the full-length AAV capsid nucleic acid sequence identified herein (eg, the AVlns and AV2cas primers). Optionally, a set according to the invention can further contain two or more additional sets of 5- and 3' primers as described here, and/or PCR probes. These primers and probes are used according to the invention to amplify signature areas of each AAV serotype, for example by bmk of quantitative PCR.

The invention further provides a kit that is useful for identifying an AAV serotype detected according to the method of the invention and/or for distinguishing new AAV from known AAV. Such a kit may further include one or more restriction enzymes, standards for AAV serotypes that provide their "signature restriction enzyme digestion assays" and/or other means to determine the serotype of the detected AAV.

In addition, kits of the invention may include instruments, a negative and/or positive control, containers, diluents and buffers for the sample, indicator charts for signature comparisons, disposable gloves, decontamination instruments, applicator sticks or containers, and sample preparation lids, as well as any desired reagents. including media, washing reagents and concentration reagents. Such reagents may be readily selected from those described herein and from conventional concentration reagents. In a desired embodiment, the washing reagent is an isotonic saline buffered to physiological pH, for example phosphate buffered saline (PBS), the elution reagent is PBS containing 0.4 M NaCl and the concentration reagent and devices. For example, one skilled in the art will recognize that reagents such as polyethylene glycol (PEG) or NH 4 SO 4 may be useful, or that devices such as filter devices, for example a filter device with a 100 K membrane, will concentrate rAAV.

The kits provided according to the invention are usable for carrying out the methods described here and for studying the biodistribution, epidemiology, transmission mode of new AAV serotypes in humans and NHPs.

Thus, the method and kits according to the invention allow the detection, identification and isolation of viral target sequences and in particular integrated viral sequences. The methods and kits are particularly suitable for use in the detection, identification and isolation of AAV sequences and which may include new AAV serotypes.

In a particular example, the method according to the invention facilitated the analysis of AAV sequences cloned by the inventors and which revealed heterogeneity of proviral sequences between cloned fragments from different animals, all distinct from the known six AAV serotypes, with the majority of the variation localized to hypervariable regions of the capsid protein . Surprising divergence from AAV sequences was noted in clones isolated from single tissue sources such as the lymph node, from an individual rhesus monkey. This heterogeneity is best explained by the apparent evolution of the AAV sequence in the individual animal due in part to extensive homologous recombination between a limited number of co-infecting parenteral vectors. These studies suggest sequence evaluation of widely disseminated vims during the course of a natural AAV infection that presumably leads to the formation of swarms of quasispecies that differ from each other in the patterns of capsid hypervariable regions. This is the first example of rapid molecular evolution of a DNA vims in a manner previously thought to be limited to RNA vims.

Sequences of several new AAV serotypes that have been identified by the method according to the invention are provided as well as the characterization of these serotypes.

Ul. New AAV serotypes

A. Nucleic acid sequences

Nucleic acid sequences of new AAV serotypes that were identified by the method of the invention are provided here, see SEQ ID no. 1, 9 - 59 and 117 - 120 to which reference is made. Reference must also be made to Figure 1 and the sequence lists.

For the new serotype AAV7, the full-length sequences including the AAV5' ITRs, capsid, rep and AAV3' ITRs are provided in SEQ ID NO: 1.

For other new AAV serotypes according to the invention, a roughly 3.1 kb fragment that was isolated according to the method of the invention is provided. This fragment contains sequences encoding the full-length capsid protein or all or part of the sequences encoding the rep protein. These sequences include the clones identified below.

For further other AAV serotypes, the signature region encoding the capsid protein is provided. For example, the AAV10 nucleic acid sequences of the invention include those illustrated in Figure 1 [see SEQ ID No. 117 which spans 255 bases].

The AAV11 nucleic acid sequences according to the invention include the DNA sequences illustrated in Figure 1 [see SEQ ID No. 118 which spans 258 bases]. The AAV 12 nucleic acid sequences according to the invention include the DNA sequences illustrated in figure 1 [see SEQ ID no. 19 which consists of 258 bases]. Using the methodology as described above, the AAV10, AAV11 and AAV12 sequence can further be easily identified and used for a number of purposes including that described for AAV7 and other new serotypes according to the invention.

Figure 1 provides the non-human primate (NHP) AAV nucleic acid sequences of the invention in an arrangement with all previously published AAV serotypes, AAV1 [SEQ ID No. 6], AAV2 [SEQ ID No. 7] and AAV3 [SEQ ID No. 8 ]. These new NHP sequences include those given in the following Table 1 and which are identified by clone no.:

A new NHP clone was generated by splicing capsid fragments from two chimpanzee adenoviruses into an AAV2 rep construct. The new clone, A3,1, is also named Ch.5 [SEQ ID No. 20]. In addition, the invention includes two human AAV sequences called H6 [SEQ ID No. 25] and H2 [SEQ ID No. 26].

The AAV nucleic acid sequences according to the invention further comprise the strand that is complementary to the strands provided in the sequence as given in Figure 1 and in the sequence lists [SEQ ID no. 1,9-59, 117-120], nucleic acid sequences as well as RNA and cDNA sequences corresponding to the sequences given in Figure 1 and the sequence list [SEQ ID No. 1,9-59, 117-120], and their complementary strands. Also included in the nucleic acid sequences according to the invention are natural variants and engineered modifications of the sequences according to figure 1 and the sequence lists [SEQ ID no. 1,9-59, 117-120] and their complementary strands. Such modifications include, for example, tags well known in the art, methylation, and substitution of one or more of the naturally occurring nucleotides with a degenerate nucleotide.

Also included in the invention are nucleic acid sequences that are more than 85%, preferably at least around 90% and most preferably at least around 95%, especially at least around 98 to 99% identical or homologous to the sequences according to the invention including figure 1 and the sequence list [SEQ ID no. 1, 9 - 59, 117-120]. These terms are as defined herein.

Also included in the invention are fragments of the new AAV sequences identified by the methods described here. Suitable fragments are at least 15 nucleotides long and include functional fragments, i.e. fragments that are of biological interest. In one embodiment, these fragments are such of the new sequences with Figure 1 and the sequence lists [SEQ ID no. 1,9-59, 117-120], their complementary strands, cDNA and RNA that are complementary thereto.

Examples of suitable fragments are provided with a view to the location of these fragments on AAV1, AAV2 or AAV7. However, the person skilled in the art, using the devices provided here (achieved using the Clustal W program at default settings), or similar techniques for generating a device with other new serotypes according to the invention, can easily identify the exact nucleotide start and stop -codons for desired fragments.

Examples of suitable fragments include the sequences encoding the three variable proteins (vp) of the AAV capsid that are alternative splice variants: vpl [eg, nt 825 to 3049 of AAV7, SEQ ID NO: 1]; vp2 [eg nt 1234 - 3049 of AAV7, SEQ ID NO: 1]; and vp3 [for example, nt 1434 - 3049 of AAV7, SEQ ID NO: 1]. It is worth noting that AAV7 has an unusual GTG start codon. Apart from a few housekeeping genes, such a start codon has not previously been reported in DNA vimser. The start codons for vpl, vp2 and vp3 for other AAV serotypes are thought to be such that they allow the cellular machinery in the host cell in which they reside to produce vpl, vp2 and vp3 in a ratio of 10%: 10%:80%, respectively, to allow efficient assembly of the virion. However, the AAV7 virion has been found to assemble efficiently even with this rare GTG start codon. Thus, the inventors assume that it is desirable to change the vp3 start codon of other AAV serotypes to contain this rare GTG start codon to improve packaging efficiency, to change the virion structure and/or to change the location of epitopes (eg neutralizing antibody epitopes) of other AAV serotypes. The start codons can be changed by bmk by conventional techniques including, for example, site-directed mutagenesis. Thus, the present invention includes modified AAV virions of any selected serotype, consisting of a vp3, and/or optionally vpl and/or vp2 with start codons changed to GTG.

Other suitable fragments of AAV including a fragment containing the start codon for the AAV capsid protein [for example, nt 468 to 3090 of AAV7, SEQ ID NO: 1, nt 725 to

3090 of AAV7, SEQ ID No. 1 and corresponding regions of other AAV serotypes]. Additional other fragments of AAV7 and the other new AAV serotypes identified using the methods described herein include those encoding the rep proteins, including rep 78 [eg, initial codon 334 of Figure 1 for AAV7], rep 68 [initial codon nt 334 of

Figure 1 for AAV7], rep 52 [initial codon 1006 of Figure 1 for AAV7] and rep 40 [initial codon 1006 of Figure 1 for AAV7]. Other fragments of interest may include the AAV5' inverted terminal repeat ITRs [nt 1 to 107 of Figure 1 for AAV7]; AAV3'ITRs [nt 4704 to 4721 of Figure 1 for AAV7], P19 sequences, AAV P40 sequences, rep binding site and terminal resolute site (TRS). Further other suitable fragments will be readily apparent to those skilled in the art. The suitable regions in other new serotypes according to the invention can be easily determined with reference to Figure 1 or by using conventional alignment techniques with the sequences provided here.

In addition to including the nucleic acid sequences given in the figures and the sequence list, the invention includes nucleic acid molecules and sequences designed to express the amino acid sequences, proteins and peptides of the AAV serotypes according to the invention. Thus, the invention includes nucleic acid sequences that encode the following new AAV amino acid sequences: Cl [SEQ ID No. 60], C2 [SEQ ID No. 61], C5 [SEQ ID No. 62], A3-3 [SEQ ID No. 66], A3-7 [SEQ ID No. 67], A3-4 [SEQ ID No. 68], A3-5 [SEQ ID No. 69], 3.3b [SEQ ID No. 62], 223.4 [SEQ ID No. 73 ], 223-5 [SEQ ID No. 74], 223-10 [SEQ ID No. 75], 223-2 [SEQ ID No. 76], 223-7 [SEQ ID No. 77], 223-6 [ SEQ ID No. 78], 44-1 [SEQ ID No. 79], 44-5 [SEQ ID No. 80], 44-2 [SEQ ID No. 81], 42-15 [SEQ ID No. 84] , 42-8 [SEQ ID No. 85], 42-13 [SEQ ID No. 86], 42-3 A [SEQ ID No. 87], 42-4 [SEQ ID No. 88], 42-5A [ SEQ ID No. 89], 42-1B [SEQ ID No. 90], 42-5B [SEQ ID No. 91], 43-1 [SEQ ID No. 92], 43-12 [SEQ ID No. 93] , 43-5 [SEQ ID No. 94], 43-21 [SEQ ID No. 96], 43-25 [SEQ ID No. 97], 43-20 [SEQ ID No. 99], 24.1 [SEQ ID No. 101], 42.2 [SEQ ID No. 102], 7.2 [SEQ ID No. 103], 27.3 [SEQ ID No. 104], 16.3 [SEQ ID No. 105], 42,10 [SEQ ID No. 106], 42-3B [SEQ ID No. 107], 42-11 [SEQ ID No. 108], Fl [SEQ ID No. 10 9], F5 [SEQ ID No. 110], F3 [SEQ ID No. 111], 42-6B [SEQ ID No. 112] and/or 42-12 [SEQ ID No. 113] and artificial AAV serotypes that are generated using these sequences and/or unique fragments thereof.

As used herein, artificial AAV serotypes include without limitation AAV with a non-naturally occurring capsid protein. Such an artificial capsid can be generated by any suitable technique using a new AAV sequence according to the invention (for example a fragment of a vpl capsid protein) in combination with heterologous sequences that can be obtained from another AAV serotype (known or new ), non-contiguous parts of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype can be, without limitation, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

B. AAV amino acid sequences, proteins and peptides

The invention provides proteins and fragments thereof encoded by the nucleic acid sequence of the novel AAV serotypes identified herein including, for example, AAV7 [nt 825 to 3049 of AAV7, SEQ ID No. 1], the other novel serotypes as described herein. Thus, the capsid proteins of the new serotypes according to the invention, including: H6 [SEQ ID No. 25], H2 [SEQ ID No. 26], 42-2 [SEQ ID No. 9], 42-8 [SEQ ID No. 27 ], 42-15 [SEQ ID No. 28], 42-5b [SEQ ID No. 29], 42-lb [SEQ ID No. 30], 42-13 [SEQ ID No. 31], 42-3a [ SEQ ID No. 32], 42-4 [SEQ ID No. 33], 42-5a [SEQ ID No. 34], 42-10 [SEQ ID No. 35], 42-3b [SEQ ID No. 36] , 42-11 [SEQ ID No. 37], 42-6b [SEQ ID No. 38], 43-1 [SEQ ID No. 39], 43-5 [SEQ ID No. 40], 43-12 [SEQ ID No. 41], 43-20 [SEQ ID No. 42], 43-21 [SEQ ID No. 43], 43-23 [SEQ ID No. 44], 43-25 [SEQ ID No. 45], 44.1 [SEQ ID No. 47], 44.5 [SEQ ID No. 47], 223.10 [SEQ ID No. 48], 223.2 [SEQ ID No. 49], 223.4 [SEQ ID No. 50], 223.5 [SEQ ID No. .51], 223.6 [SEQ ID No. 52], 223.7 [SEQ ID No. 53], A3.4 [SEQ ID No. 54], A3.5 [SEQ ID No. 55], A3.7 [SEQ ID No. 56], A3.3 [SEQ ID No. 57], 42.12 [SEQ ID No. 58] and 44.2 [SEQ ID No. 59], are readily generated using conventional techniques from the open reading frames provided for the above r listed clones.

The invention further encompasses AAV serotypes which are generated using sequences of the new AAV serotypes according to the invention and which are generated using synthetic, recombinant or other techniques well known to those skilled in the art. The invention is not limited to new AAV amino acid sequences, peptides and proteins that are expressed from the new AAV nucleic acid sequences according to the invention and includes amino acid sequences, peptides and proteins that are generated by other methods known in the art, including for example by chemical synthesis, or by other synthetic techniques, or by other methods. For example, the sequences of any one of C1 [SEQ ID NO: 60], C2 [SEQ ID NO: 61], C5 [SEQ ID NO: 62], A3-3 [SEQ ID NO: 66], A3- 7 [SEQ ID No. 67], A3-4 [SEQ ID No. 68], A3-5 [SEQ ID No. 69], 3.3b [SEQ ID No. 62], 223.4 [SEQ ID No. 73], 223-5 [SEQ ID No. 74], 223-10 [SEQ ID No. 75], 223-2 [SEQ ID No. 76], 223-7 [SEQ ID No. 77], 223-6 [SEQ ID No. 78], 44-1 [SEQ ID No. 79], 44-5 [SEQ ID No. 80], 44-2 [SEQ ID No. 81], 42-15 [SEQ ID No. 84], 42 -8 [SEQ ID No. 85], 42-13 [SEQ ID No. 86], 42-3A [SEQ ID No. 87], 42-4 [SEQ ID No. 88], 42-5A [SEQ ID No. .89], 42-1B [SEQ ID No. 90], 42-5B [SEQ ID No. 91], 43-1 [SEQ ID No. 92], 43-12 [SEQ ID No. 93], 43- 5 [SEQ ID No. 94], 43-21 [SEQ ID No. 96], 43-25 [SEQ ID No. 97], 43-20 [SEQ ID No. 99], 24.1 [SEQ ID No. 101] , 42.2 [SEQ ID No. 102], 7.2 [SEQ ID No. 103], 27.3 [SEQ ID No. 104], 16.3 [SEQ ID No. 105], 42.10 [SEQ ID No. 106], 42-3B [ SEQ ID No. 107], 42-11 [SEQ ID No. 108], Fl [SEQ ID No. 109], F5 [SEQ ID No. 110], F3 [SEQ ID No. 111], 42-6 B [SEQ ID NO: 112], and/or 42-12 [SEQ ID NO: 113] are readily generated using a number of techniques.

Suitable production techniques are well known to those skilled in the art, see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press (Cold Spring Harbor, NY). Alternatively, peptides can also be synthesized by the well-known solid phase peptide synthesis method (Merrifield, "J. Am. Chem. Soc", 85:2149 (1962); Stewart and Young, "Solid PhasePeptide Synthesis" (Freeman, San Francisco, 1969), p. 27-62). These and other suitable production methods are within the expert's area of knowledge and are not a limitation of the invention.

Particularly desirable proteins include the AAV capsid proteins encoded by the nucleotide sequences identified above. The sequences of many of the capsid proteins according to the invention are provided in a device in Figure 12 and/or in the sequence lists, SEQ ID No. 2 and 60 to 115 to which reference is made when it comes to details. The AAV capsid consists of three proteins, vpl, vp2 and vp3, which are alternative splice variants. The full-length sequence provided in these figures is that of vpl. Based on the numbering of the AAV7 capsid [SEQ ID No. 2], the sequences of vp2 span amino acids 138 - 737 of AAV7 and the sequences of vp3 span amino acids 203 - 737 of AAV7. With this information, the person skilled in the art can easily determine the location of the vp2 and vp3 proteins for the other new serotypes according to the invention.

Other desirable proteins and fragments of the capsid protein include the constant and variable regions, located between hypervariable regions (HPV) and the sequences of the HPV regions themselves. An algorithm developed to determine areas of sequence divergence in AAV2 has yielded 12 hypervariable regions (HVR) of which 5 overlap or are part of the four previously described variable regions, see Chiorini et al, "J. Virol", 73:1309-19 (1999), Rutledge et al, "J. Virol.", 72:309-319. Using this algorithm and/or the setup techniques as described here, the HVR of the new AAV serotypes is determined. With respect to numbers of AAV2 vpl [SEQ ID No. 70], for example, the HVR is located as follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264, HVR4, aa 381-383, HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa 500-504; HVR8, aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594; HVR11, aa 658-667; andHVR12, aa 705-719. By using a device produced according to conventional methods and the new sequences given here, see for example Figure 2, the location of this HVR in the new AAV serotypes according to the invention can be easily determined. Using, for example, Figure 2, one can easily determine that for AAV7 [SEQ ID no. 2] HVR1 is located at aa 146 - 152; HVR2 is located at 182-187; HVR3 is located at 263-266, HVR4 is located at aa 383-385, HVR5 is located at aa 451-475; HVR6 is located at aa 491-496, HVR7 is located at aa 501-505; HVR8 is located at aa 513-521; isHVR9 located at 533-554; isHVRlO located at aa 583-596; HVR1 1 is located at aa 660-669, HVR12 is located at 707-721. Using the information provided here, the HVRs for the other new serotypes according to the invention can be easily determined.

In addition, in the capsid, the amino acid identity cassettes are identified. These cassettes are of particular interest as they are useful in the construction of artificial serotypes, for example by replacing an HVR1 cassette of a selected serotype with an HVR1 cassette of another serotype. Some of these identity cassettes are indicated in Figure 2, see Figure 2, which provides the Clustal X device which has a ruler shown below the sequence and which starts at 1 for the position of the first residue. The line above ruler one is used to mark highly conserved positions. The characters (<*>, :,.) are used. "<*>" suggests positions that have a single, fully conserved residue.":" suggests that a "strong" group is fully conserved."." indicates that a "weaker" group is fully conserved. These are all positively judging groups that appear in the Gonnet Pam250 matrix. The strong groups are defined as a strong assessment >0.5 and the weak groups are defined as a weak assessment <0.5.

In addition, examples of other suitable fragments of AAV capsids include, for the purpose of numbering AAV2 [SEQ ID No. 70], aa 24-42, aa 25-28; aa 81 - 85; Aal33-165; aa 134-165; aa 137-143; aa 154-156; aa 194-208; aa 261-274; aa262-274; aa 171-173; aa 413-417; aa 449-478; aa 494-525; aa 534-571; aa 581-601; aa 660-671; aa 709-723. Additional other desirable fragments include, for example, in AAV7, amino acids 1 to 184 of SEQ ID NO: 2, amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724 to 736; aa 185 to 198; aa 260 to 273; aa 447 to 477; aa 495 to 602; aa 660 to 669; and aa 707 to 723. Additional other desirable regions, based on the numbering of AAV7 [SEQ ID NO: 2] are selected from the group consisting of aa 185 to 198; aa 260 to 273; aa 447 to 477; aa 495 to 602; aa 660 to 669; and aa 707 to 723. By using the device provided here, obtained by using the Clustal X program at default settings, or by using other commercially or commonly available device programs at default settings, the person skilled in the art can easily determine corresponding fragments of the new AAV capsids according to the invention.

Other desirable proteins are the AAV rep proteins [aa 1 to 623 of SEQ ID No. 3 for AAV7] and functional fragments thereof including, for example, aa 1 to 171, aa 172 to 372, aa 373 to 444, aa 445 to 623 of SEQ ID No. 3, among others. Appropriately, these fragments are at least 8 amino acids long, see Figure 3. Comparable regions can be identified in the proteins of other new AAVs according to the invention using the techniques described here and those known in the art. In addition, fragments of other desired lengths can easily be used. Such fragments can be produced recombinantly or by other suitable means, for example chemical synthesis.

The sequences, proteins and fragments of the invention may be prepared by any suitable means including recombinant production, chemical synthesis or other synthetic means. Such production methods are within the expert's area of knowledge and are not a limitation of the invention.

IV. Production of rAAV with new AAV capsids

The invention includes new, wild-type AAV serotypes that have been identified according to the invention, where the sequences of these wild-type AAV serotypes are free of DNA and/or cellular material with which these viruses are associated in nature. In another aspect, the invention provides molecules that use the new AAV sequences according to the invention, including fragments thereof, for the production of molecules that are usable when delivering a heterologous gene or other nucleic acid sequences to a target cell.

The molecules according to the invention which contain sequences of a new AAV serotype according to the invention include any genetic element (vector) which can be delivered to a host cell, for example naked DNA, a plasmid, a phage, a transposon, a cosmid, a episome, a protein in a non-viral delivery vehicle (eg, a lipid-based vehicle), a vims, and so on that transfers the sequence carried thereon. The selected vector can be delivered by any suitable method including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated pellets, viral infection and protoplast fusion. The methods used to construct any embodiment of the invention are well known to those skilled in the art of nucleic acid re-engineering and include genetic engineering, recombinant engineering and synthetic techniques, see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, N. Y.

In one embodiment, the vectors according to the invention contain sequences that encode a new AAV capsid according to the invention (for example AAV7 capsid, AAV 44-2 (rh.10), an AAV10 capsid, an AAV11 capsid, an AAV12 capsid), or a fragment of a or more of these AAV capsids. Alternatively, the vectors themselves may contain the capsid protein or a fragment thereof.

Optionally, the vectors according to the invention may contain sequences that encode AAV rep proteins. Such rep sequences can be from the same AAV serotype that provides the cap sequence. Alternatively, the invention provides vectors where the rep sequence is from an AAV serotype that differs from that which provides the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (eg, separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are expressed from the same source as the cap sequences. In this embodiment, the rep sequences can be fused in frame to the cap sequences of a different AAV serotype to form a chimeric AAV vector. Optionally, the vectors according to the invention can further contain a minigene comprising a selected transgene which is flanked by the AAV5' ITR and

AAV3'ITR.

Thus, in one embodiment, the vectors described here contain nucleic acid sequences that encode an intact AAV capsid which may be from a single AAV serotype (for example AAV7 or another new AVV). Alternatively, these vectors contain sequences encoding artificial capsids containing one or more fragments of AAV7 (or another novel AAV capsid) fused to heterologous AAV or non-AAV capsid proteins or fragments thereof. These artificial capsid proteins are selected from non-contiguous portions of the AAV7 (or another new AAV) capsid or from capsids of other AAV serotypes. For example, it may be desirable to modify the coding regions of one or more of the AAV vpl, for example in one or more of the hypervariable regions (ie HPV1-12), or vp2 and/or vp3.1 another example may it may be desirable to change the start codon for the vp3 protein to GTG. These modifications can occur to increase expression, yield and/or improve purification in the chosen expression systems, or for another desired purpose (for example to change the tropism or change the neutralizing antibody epitopes).

The vectors described here, for example a plasmid, are usable for a number of purposes but are particularly well suited for use in the production of an rAAV containing a capsid comprising AAV sequences or a fragment thereof. These vectors, including rAAV, their elements, construction and application are described in detail here.

In one aspect, the invention provides a method for generating a recombinant adeno-associated virus (AAV) with an AAV serotype 7 (or other novel AAV) capsid, or a portion thereof. One such method involves culturing a host cell containing a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype 7 (or other novel AAV) capsid protein or fragment thereof, as defined herein; a functional rep gene; a minigene consisting of a minimum of AAV inverted terminal repeaters (ITRs) and a transgene; and sufficient helper functions to allow packaging of the minigene into the AAV7 (or another new AAV) capsid protein.

The components required for cultivation in the host cell for packaging of an AAV minigene into an AAV capsid can be provided in the host cell in trans. Alternatively, any one or more of the required components (eg, minigene, rep sequences, cap sequences, and/or helper functions) can be provided by a stable host cell engineered to contain one or more of the required components using i and methods known to the person skilled in the art. Preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with the transgene. In yet another alternative, a selected stable host cell may contain one or more selected components under the control of a constitutive promoter and one or more other selected components under the control of one or more inducible promoters. For example, a stable host cell can be generated derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Additional other stable host cells can be generated by one skilled in the art.

The minigene, the rep sequences, the cap sequences and the helper functions necessary to produce the rAAV of the invention can be delivered to the packaging host cell in the form of any genetic element that transmits the sequences carried thereon. The selected genetic element may be delivered by any suitable method including those described herein. The methods used to construct any embodiment of the invention are well known to those skilled in the art of nucleic acid re-engineering and include genetic engineering, recombinant engineering and synthetic techniques, see, for example, Sambrook et al. in "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known and the choice of a suitable method is not a limitation of the invention, see for example K. Fisher et al. in "J. Virol.", 70:520-532 (1993) as well as US 5,478,745.

A. Minigen

The minigene consists as a minimum of a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeaters (ITRs). It is this minigene that is packaged into a capsid protein and delivered to a selected host cell.

1. Transgenic

The transgene is a nucleic acid sequence, heterologous to the vector sequences flanking the transgene, which encodes a polypeptide, protein or other product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transgenic transcription, translation and/or expression in a host cell.

The composition of the transgene sequence will depend on the application for which the resulting vector will be used. For example, one type of transgene sequence includes a reporter sequence that upon expression produces a detectable signal. Such reporter sequences include without limitation DNA sequences encoding β-lactamase, β-galactosidase

(LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which there is a high-affinity antibody directed against it or can be produced by conventional means, and fusion proteins comprising a membrane-bound protein which is suitably fused to an antigen tag domain from, among other things, hemagglutinin or Mye.

These coding sequences, associated with regulatory elements that drive their expression, provide signals detectable by conventional means including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent-activating cell sorting assays, and immunological assays, including enzyme-linked immunoabsorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. Where, for example, the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by analyzes of β-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be measured visually by color or light production in a luminometer.

However, the transgene is preferably a non-marker sequence that encodes a product useful in biology and medicine such as proteins, peptides, RNA, enzymes, or catalytic RNAs. Desired RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs and antisense RNAs. An example of a usable RNA sequence is a sequence which deletes expression of a target nucleic acid sequence in the treated animal.

The transgene can be used to correct or improve gene defects and that can include defects where normal genes are expressed at less than normal levels or defects where the functional gene product is not expressed. A preferred type of transgene sequences encodes a therapeutic protein or polypeptide that is expressed in a host cell. The invention further includes the use of multiple transgenes, for example to correct or improve a gene defect caused by a multisubunit protein. In certain situations, a different transgene can be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large, for example for an immunoglobulin, the platelet-derived growth factor, or a dystrophin protein. In order for the cell to produce the multisubunit protein, the cell is infected with the recombinant virus containing each of the different subunits. Alternatively, different subunits of a protein may be encoded by the same transgene. In this case, a single transgene includes the DNA encoding each of the subunits with DNA for each subunit separated by an internal ribozyme entry site (IRES). It is desirable when the size of the DNA encoding each of the subunits is small, for example that the total size of the DNA encoding the subunits and the IRES is less than five kilobases. As an alternative to an IRES, the relevant DNA can be separated by sequences encoding a 2A peptide, which is self-cleaving in a post-translational event, see for example M L. Donnelly et al. in "J. Gen. Virol.", 78(Pt 1): 13-21 (Jan 1997); S Fourier et al. in "Gene Ther.", 8(ll):864-873 (June 2001); H Klump et al. in "Gene Ther.", 8(10:811-817 (May 2001). This 2A peptide is significantly smaller than an IRES, making it suitable for use when space is a limiting factor. However, the selected transgene may encode a any biologically active product or other product, for example a product desirable for studies.

Suitable transgenes can be easily selected by the person skilled in the art. The choice of transgene is not considered a limiting factor for the invention.

2. Regulatory elements

In addition to the major elements identified above for the minigene, the vector also includes necessary conventional control elements operably linked to the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced according to the invention. As used herein, "operably linked" sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

Expression control sequences include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals, sequences that stabilize cytoplasmic mRNAs; sequences that improve translational efficiency (that is, the Kozak consensus sequence); sequences that improve protein stability; and, if desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences, including promoters that are native, constitutive, inducible and/or tissue specific, are well known in the art and can be used.

Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), [see, for example, Boshart et al. in "Cell", 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-active promoter, the phosphoglycerol kinase (PGK) promoter, and the EF la promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, for example acute phase, a particular differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from a number of commercial sources including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems are described and can be easily selected by the person skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible ovine metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system [WO 98/10088]; the ecdysone insect promoter [No et al. "Proe. Nati. Acad. Sei. USA", 93:3346-3351 (1996)], the tetracycline-repressible system [Gossen et al., "Proe. Nati. Acad. Sei. USA", 89:5547-5551] ( 1992)], the tetracycline-inducible system [Gossen et al. "Science", 268:1766-1769 (1995), see also Harvey et al. "Curr. Opin. Chem. Biol.", 2:512-518 (1998)], the RU486-inducible system [Wang et al., "Nat. Biotech.", 15:239-243 (1997) and Wang et al. al., "Gene Ther.", 4:432-441 (1997)] and the rapamycin-inducible system [Magari et al., "J. Clin. Invest", 100:2865-2872 (1997)]. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological condition, for example temperature, acute phase, a particular differentiation state of the cell or only in replicating cells.

In another embodiment, the native promoter for the transgene will be used. The native promoter may be preferred when it is desired that the expression of the transgene

mimics the native expression. The native promoter can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific mode, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements such as enhancer elements, polyadenylation sites or Kozak consensus sequences can also be used to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linked to a tissue-specific promoter. If, for example, expression in the gill muscle is desired, a promoter that is active in muscles should be used. These include promoters from genes encoding skeletal P-actin, myosin light chain 2A, dystofin, muscle - creatine kinase, as well as synthetic muscle promoters that activate higher than naturally occurring promoters, (see Li et al. in "Nat. Biotech.", 17:241-245 (1999)). Examples of promoters that are tissue specific are known for the liver (albumin, Miyatake et al. in "J. Virol.", 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al. in "Ge-ne Ther. ", 3:1002-9 (1996); α-fetoprotein (AFP); Arbuthnot et al. in "Hum. Gene Ther.", 7:1503-14 (1996)), benoosteocalcin (Stein et al. in "Mol . Biol. Rep." 24:185-96 (1997)), benzyloprotein (Chen et al. in "J. Bone Miner. Res.", 11:654-64 (1996)), lymphocytes (CD2, Hansal et al . in "J. Immunol.", 161:1063-8 (1998), immunoglobulin heavy chain; T cell receptor a chain), neuronal as neuron-specific enolase (NSE) promoter (Andersen et al. in "Cell. Mol . Neurobiol.", 13:503-15 (1993)), neurofilament light-chain gene (Piccioli et al. in "Proe. Nati. Acad. Sei. USA", 88:5611-5 (1991)) and the neuro -specific vgf gene (Piccioli et al. in "Neuron", 15:373-84 (1995)), among others.

Optionally, plasmids carrying therapeutically useful transgenes may also include selectable markers or reporter genes may include sequences encoding geneticin, hygromycin or purimycin resistance, among others. Such selectable reporters or marker genes (preferably located outside the viral genome to be provided via the method of the invention) can be used to signal the presence of the plasmids in bacterial cells, such as ampicillin resistance. Other components of the plasmid may include an origin of replication. The choice of these and other promoters and vector elements is conventional and many such sequences are available, see for example Sambrook et al. and the references stated therein.

The combination of the transgene, promoter/enhancer, and the 5 and 3' ITRs is referred to as a "minigene" for convenience in the present description. Provided together with the teachings according to the invention, the construction of such a minigene will be easily accomplished by resorting to conventional techniques.

3. Delivery of the minigene to a packaging host cell

The minigene can be carried on any suitable vector, for example a plasmid, which is delivered to a host cell. Plasmids which are usable according to the invention can be constructed so that they are suitable for replication and possibly integration in prokaryotic cells, mammalian cells or both. These plasmids (or other vectors carrying the 5' AAV ITR-heterologous molecule-3' ITR) contain sequences that allow replication of the minigene in eukaryotes and/or prokaryotes and selection markers for these systems. Selectable markers or reporter genes may include sequences encoding geneticin, hygromycin, or purimycin resistance, among others. The plasmids may also contain certain selectable reporters or marker genes that can be used to signal the presence of the vector in bacterial cells, such as ampicillin resistance. Other components of the plasmid may include an origin of replication and an amplicon, for example the amplicon system using the Epstein Barr virus nuclear antigen. This amplicon system, or other similar amplicon components, allows high copy episomal replication in the cells. Preferably, the molecule carrying the minigene is transfected into the cell where it can exist transiently. Alternatively, the minigene (carrying the relevant 5' AAV ITR heterologous molecule-3' ITR) may be stably integrated into the genome of the host cell, either chromosomally or as an episome. In certain embodiments, the minigene may be present in multiple copies, possibly in head-to-head, head-to-tail or tail-to-tail contact mers. Suitable transfection techniques are well known and can be easily used to deliver the minigene to the host cell.

Upon delivery of the vector comprising the minigene by transfection, the vector is generally delivered in an amount of from about 5 µg to about 100 µg DNA and preferably about 10 to about 50 µg DNA to about 1 x 10<4> cells to about 1 x 1013 cells and preferably around 10<5> cells. However, the relative amounts of vector DNA in relation to host cells can be adjusted, taking into account factors such as the chosen vector, delivery method and the chosen host cells.

B. Rep and Cap sequences

In addition to the minigene, the host cell contains the sequences that drive the expression of the new AAV capsid protein (for example AAV7 or another new AAV capsid or an artificial capsid protein comprising a fragment of one or more of these capsids) in the host cell and rep sequences of the same serotype as the serotype of the AAV ITRs found in the minigene. The AAV cap and -rep sequences can be independently obtained from an AAV source as described above and can be introduced into the host cell in any manner known to the person skilled in the art as described above. In addition and when pseudotyping a new AAV capsid according to the invention, the sequences that encode each of the essential rep proteins can be supplemented with the same AAV serotype, or the sequences that encode the rep proteins can be supplemented with different AAV serotypes (for example AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 or one of the new serotypes identified here). For example, the rep78/68 sequences may be from AAV2 while the rep52/40 sequences may be from AAV1.

In one embodiment, the host cell contains stable capsid protein under the control of a suitable promoter, such as those described above. Most preferably, and in this embodiment, the capsid protein is expressed under the control of an inducible promoter. In another embodiment, the capsid protein is delivered to the host cell in trans. Delivered to the host cell in trans, the capsid protein can be delivered via a plasmid containing se sequences necessary to control the expression of the selected capsid protein in the host cell. Preferably, the plasmid which carries the capsid protein, when delivered to the host cell in trans, also carries other sequences which are necessary for packaging the rAAV in question, for example the rep sequences.

In another embodiment, the host cell stably contains the rep sequences under the control of a suitable promoter such as those described above. Preferably, the essential rep proteins, in this embodiment, are expressed under the control of an inducible promoter. In another embodiment, the rep proteins are provided to the host cells in trans. Delivered to the host cell in trans, the rep proteins can be delivered via a plasmid that contains the sequences necessary to control the expression of the selected rep proteins in the host cell. Most preferably, the plasmid carrying the capsid, when delivered to the host cell in trans, also carries other sequences that are necessary to package the rAAV in question, for example the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences can be transfected into the host cell on a single nucleic acid molecule and exist stably in the cell as an episome. In another embodiment, the rep and cap sequences are stably integrated into the genome of the cell. Another embodiment has the rep and trans sequences transiently expressed in the host cell. For example, a usable nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, any spacer arranged between the promoter and the starting site for the rep gene sequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences can be provided on a vector that contains other DNA sequences to be introduced into the host cell. For example, the vector may contain the rAAV construct comprising the minigene. The vector may comprise one or more genes encoding the helper functions, for example the adenoviral proteins E1, E2a, and E40RF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct can be any of the constitutive, inducible or native promoters well known to those skilled in the art or as discussed above. In one embodiment, an AAV P5 promoter sequence is used. The choice of the AAV to provide any of these sequences is not a limitation of the invention.

In another preferred embodiment, the promoter for rep is an inducible promoter as discussed above in connection with the transgene regulatory elements. A preferred promoter for reexpression is the T7 promoter. The vector comprising the rep gene regulated by the T7 promoter and the cap gene is transfected or transformed into a cell which either constitutively or inducibly expresses the T7 polymerase, see WO 98/10088, published March 12, 1998.

The spacer is an optional element in the construction of the vector. The spacer is a DNA sequence that is placed between the promoter and the rep gene ATG start site. The spacer can have any desired construction, i.e. it can be an arbitrary sequence of nucleotides or can optionally encode a gene product, for example a marker gene. The spacer may contain genes that typically incorporate start/stop and polyA sites. The spacer can be a non-coding DNA sequence from a prokaryote or a eukaryote, a repetitive non-coding sequence, a coding sequence without the transcription controls or a coding sequence with the transcription controls. Two example sources for the spacer sequence are a phage ladder sequence or the yeast ladder sequence, which are commercially available, for example from Gibco or Invitrogen, among others. The spacer can be of any size sufficient to reduce expression of the rep78 and rep68 gene products and leave the rep52, rep40 and cap gene products expressed at normal levels. The length of the spacer can therefore be from around 10 bp to around 10.0 kbp, preferably in the worm mode around 100 bp to around 8.0 kbp. In order to reduce the possibility of recombination, the spacer preferably has a length shorter than 2 kbp, however, the invention is not limited by this.

Although the molecule or molecules providing rep and cap may exist in the host cell transiently (that is, by transfection), it is preferred that one or both of the rep and cap proteins and the promoter or promoters controlling their expression are stably expressed in the host cell, for example as an episome or by integration into the chromosome of the host cell. The methods used to construct embodiments of the invention are conventional genetic engineering or recombinant engineering techniques such as those described in the above references. While the present description provides illustrative examples of the specific constructs, the person skilled in the art using the information provided here will be able to choose to construct any other suitable constructs using a selection of spacers, P5 promoters and other elements, including at least one translational start and stop signal, and the eventual addition of polyadenylation sites.

In another embodiment of the invention, the rep or cap protein can be provided stably by a host cell.

C. Auxiliary functions

The packaging host cell also requires helper functions to package the rAAV of the invention. These functions can possibly be provided by a herpes virus. Preferably, the necessary helper functions are all provided from a human or non-human primate adenovirus source such as those described above and/or which are available from a number of sources including the American Type Culture Collection (ATCC), Manassas, VA ( US). In a currently preferred embodiment, the host cell is provided with and/or contains an Ela gene product, an Elb gene product, an E2a gene product and/or an E4 ORF6 gene product. The host cell may contain other adenoviral genes such as VAI RNA, but these genes are not necessary. In a preferred embodiment, no other adenovirus genes or

-gene functions present in the host cell.

By "adenoviral DNA expressing the Ela gene product" is meant any adenoviral sequence encoding Ela or any functional Ela part. Adenoviral DNA expressing E2a gene product and adenoviral DNA expressing E4 ORF6 gene product are defined in a similar way. Also included are any alleles or other modifications of the adenoviral gene or functional portions thereof. Such modifications can be intentionally introduced by resorting to conventional genetic engineering or mutagenic techniques to somehow increase adenoviral function as well as naturally occurring allelic variants thereof. Such modifications and methods of manipulating DNA to enhance these adenovirus gene functions are well known to those skilled in the art.

Adenovirus Ela, -Elb, -E2a, and/or E40RF6 gene product, as well as any other desired helper functions, can be provided in any manner that allows their expression in a cell. Each of the sequences encoding these products may be on a separate vector, or one or more genes may be on the same vector. The vector can be any vector known in the art or described herein including plasmids, cosmids and vims. Introduction into the host cell of the vector can be achieved by any means known in the art or as described above, including transfection, infection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated pellets, viral infection and protoplast fusion among others. One or more of the adenoviral genes can be stably integrated into the genome of the host cell, stably expressed as episomes or expressed transiently. The gene products may all be expressed transiently, on an episome or stably integrated, or some of the gene products may be stably expressed while others are transiently expressed. Furthermore, the promoters for each of the adenoviral genes can be independently selected from a constitutive promoter, an inducible promoter or a native adenoviral promoter. These promoters can be regulated via a specific, physiological state of the organism or cell (for example in differentiation states or in replicating or resting cells) or by exogenously added factors, as examples.

D. Host cells and packaging cell lines

The host cell may per se be selected from any biological organism including prokaryotic (eg bacterial) cells, and eukaryotic cells including insect, yeast and mammalian cells. Particularly desirable host cells are selected from any mammalian cells including, without limitation, cells such as A549, WEFfl, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38 HeLa or 293 cells (expressing functional adenoviral E1), Saos, C2C12 or L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including humans, monkeys, mice, rats, rabbits and hamsters. The choice of a mammalian species to provide the cells is not a limitation of the invention nor is the type of mammalian cells, for example fibroblast, hepatocyte, tumor cells and so on. The most desired cells carry no adenovirus genes other than E1, E2a and/or E4 ORF6; nor do they contain any other viral genes that could result in homologous recombination of a contaminating virus during the production of rAAV; and it is capable of infection or transfection of DNA and expression of the transfected DNA. In a preferred embodiment, the host cell is one that has rep and cap stably transfected into the cell.

A host cell that is usable according to the invention is a host cell that is stably transformed with the sequences encoding rep and cap and that is transfected with the relevant adenovirus E1-, -E2a- and -E40RF6 DNA and a construct carrying the minigene as described above. Stable rep- and/or cap-expressing cell lines such as B-50

(PCT/US98) 19463) and those described in US 5 658 785 can also be used in a similar way. Other desirable host cells contain the minimal adenoviral DNA sufficient to express E4 ORF6. Further other cell lines can be constructed using the new AAV rep and/or new AAV cap sequences according to the invention.

The production of a host cell according to the invention involves techniques such as composition of selected DNA sequences. This composition can be carried out using conventional techniques. Such techniques include cDNA and genomic clones, which are well known to those skilled in the art and described by Sambrook et al. supra, using overlapping oligonucleotide sequences of the adenovirus and AAV genomes, combined with polymerase chain reaction, synthetic methods and any other suitable methods that provide the desired nucleotide sequences.

Introduction of the molecules (such as plasmids or vims) into the host cell can also be carried out using techniques known to the person skilled in the art and as discussed in the present description. In a preferred embodiment, standard transfection techniques are used, for example CaPC^transfection or electroporation, and/or infection by hybrid adenovims/AAV vectors into the cell lines such as the human embryonic kidney cell line HEK 293 (a human kidney cell line containing functional adenovims E1 genes that provide transacting E1 proteins).

These new AAV-based vectors generated by the person skilled in the art are advantageous for gene delivery to selected host cells and gene therapy patients because no neutralizing antibodies to AAV7 have been found in the human population. Furthermore, early studies show no neutralizing antibodies in cyno monkey or chimpanzee populations and less than 15% cross-reactivity of AAV7 in rhesus monkeys, the species from which the serotype was isolated. Those skilled in the art can readily prepare other rAAV viral vectors containing the AAV7 capsid proteins provided therein by bmk by a number of techniques well known to those skilled in the art. One can simply prepare further other rAAV viral vectors containing the AAV7 sequences and the AAV capsids of another serotype. Corresponding advantages are achieved with the vectors which are based on other new AAVs according to the invention.

Thus, the expert in the field will easily understand that the AAV7 sequences according to the invention can be easily adapted for bmk in these and other viral vector systems for in vitro, ex vivo or in vivo gene delivery. Correspondingly, the person skilled in the art can easily select other fragments of the new AAV genome according to the invention for bmk in a number of rAAV and non-rAAV vector systems. Such vector systems may include, for example, lentivims, retrovims, poxvims, vaccine vims and adenoviral systems. Choice among these vector systems is not a limitation of the invention.

Thus, the invention further provides vectors generated by bmk of the nucleic acid and amino acid sequence of the new AAV according to the invention. Such vectors are useful for a number of purposes, including for delivery of therapeutic molecules and for bmk in vaccine regimens. Particularly desirable for delivery of therapeutic molecules are recombinant AAV containing capsids of the new AAVs according to the invention. These or other vector constructs containing new AAV sequences according to the invention can be used in vaccine regimens, for example for simultaneous delivery of a cytokine, or for delivery of the immunogen per se.

V. Recombinant genes and their application.

By using the techniques described here, the expert in the field can generate an rAAV with a capsid of a new serotype according to the invention, or a new capsid containing one or more fragments of an AAV serotype identified by the method of the invention. In one embodiment, a full-length capsid from a single serotype can be used, for example AAV7 [SEQ ID No. 2]. In another embodiment, a full-length capsid can be generated containing one or more fragments of a new serotype according to the invention, fused in frame with sequences from another, selected AAV serotype. For example, an rAAV may contain one or more of the new hypervariable region sequences of an AAV serotype according to the invention. Alternatively, the unique AAV serotypes according to the invention can be used in constructs containing other viral or non-viral sequences.

It will be readily apparent to the person skilled in the art that, in one embodiment, certain serotypes according to the invention will be particularly suitable for certain applications. For example, vectors based on AAV7 capsids according to the invention are particularly well suited for use in muscles, while vectors based on rh.10 (44-2) capsids according to the invention are particularly well suited for bmk in the lung. The use of such vectors is not limited and the person skilled in the art can use these vectors for delivery to other cell types, tissues or organs. Furthermore, vectors based on other capsids according to the invention can be used for delivery to these or other cells, tissues or organs.

A. Delivery of a transgene

In another aspect, the present invention provides a method for delivering a transgene to a host which involves transfecting or infecting a selected host cell with a vector generated with the sequences of the AAV of the invention. Methods of delivery are well known to those skilled in the art and are not a limitation of the invention.

In a desired embodiment, the invention provides a method for AAV-mediated delivery of a transgene to a host. The method involves the transfection or infection of a selected host cell with a recombinant viral vector containing a selected transgene under the control of sequences that direct the expression thereof and the AAV capsid proteins.

Optionally, a sample from the host can first be analyzed for the presence of antibodies against a selected AAV serotype. A number of analysis formats for the detection of neutralizing antibodies are well known to those skilled in the art. The choice of such an analysis is not a limitation of the invention, see for example Fisher et al. in "Nature Med.", 3(3):306-312 (March, 1997) as well as W.C. Manning et al. in "Human Gene Therapy", 9:477-485 (March 1, 1998). The results of this analysis can be used to determine whether AAV vector-containing capsid proteins of a particular serotype are preferred for delivery, for example in the absence of neutralizing antibodies specific for this capsid serotype.

In one aspect of this method, delivery of a vector with a selected AAV capsid protein may precede or follow delivery of a gene via a vector with a different serotype AAV capsid protein. Correspondingly, the delivery of a vector with other new AAV capsid proteins according to the invention can precede or follow the delivery of a gene via a vector with a different serotype AAV capsid protein. Thus, gene delivery via rAAV vectors can be used for repeated gene delivery to a selected host cell. Desirably, subsequently administered rAAV vectors carry the same transgene as the first rAAV vector while the subsequently administered vectors contain capsid proteins of serotypes different from the first vector. For example, if a first vector has AAV7 capsid proteins [SEQ ID NO: 2], later administered vectors may have capsid proteins selected from among other serotypes including AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV6, AAV10, AAV11, and AAV 12, or any of the other novel AAV capsids identified herein, including without limitation: A3.1, H2, H6, Cl, C2, C5, A3-3, A3-7, A3-4, A3-5, 3.3 b, 223.4, 223-5, 223-10, 223-2, 223-7, 223-6, 44-1, 44.5, 44_2, 42-15, 42-8, 42-13, 42-3A, 42- 4, 42-5A, 42-1B, 42-5B, 43-1, 43-12, 43-5, 43-21, 43-25, 43-20, 24.1, 42.2., 7.2., 27.3, 16.3, 42.10. 42-3B, 42-11, Fl, F5, F3, 42-6B, and or 42-12.

The recombinant vectors described above can be delivered to host cells according to published methods. The rAAV in question, preferably suspended in a physiologically acceptable carrier, can be administered to a human or non-human mammalian patient. Suitable carriers can be readily selected by one skilled in the art in light of the indication for which the transmission virus is being determined. For example, a suitable carrier includes saline which can be formulated with a number of buffer solutions (eg, phosphate buffered saline). Other examples of carriers are sterile saline solutions, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut or sesame oil, as well as water. The choice of carrier is not a limitation of the invention.

Alternatively, in addition to rAAV and one or more carriers, the preparations according to the invention may contain other conventional pharmaceutical ingredients such as preservatives or chemical stabilizers. Suitable examples of preservatives are chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol and parachlorophenol. Suitable chemical stabilizers are gelatin and albumin.

The viral vectors are administered in amounts sufficient to transfect the cell and to provide sufficient levels of gene transfer and expression to thereby provide a therapeutic benefit without undue adverse effects, or with medically acceptable physiological effects, as can be determined by one skilled in the art. Conventional and pharmaceutically acceptable routes of administration include, without limitation, direct delivery to the organ of choice (for example, intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral routes of administration . The administration routes can be combined if desired.

Doses of the viral vector will primarily depend on factors such as the condition of the individual to be treated, age, weight and health and can thus vary among different patients. For example, a therapeutically effective human dose of the viral vector generally ranges from about 1 ml to about 100 ml of solution containing concentrations of about 1 x 10<9> to 1 x 10<16> genomes of viral vector. A preferred human dosage may be from about 1 x 10<13> to 1 x 10<16> AAV genomes. The dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending on the therapeutic application for which the recombinant vector is used. The expression levels of the transgene can be monitored to determine the frequency of dosing resulting in viral vectors, preferably AAV vectors containing the minigene. Optionally, dosage regimens corresponding to that described for therapeutic purposes can be used for immunization using the preparations of the invention.

Examples of therapeutic products and immunogenic products for delivery via AAV-containing vectors according to the invention will be described below. These vectors can be used for a number of therapeutic or vaccine purposes as described here. In addition, these vectors can be delivered in combination with one or more other vectors or active ingredients in a desired therapeutic and/or vaccine regimen.

B. Therapeutic transgenes

Useful therapeutic products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), weight hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any of the transforming growth factor P superfamily including TGF P, activins, inhibins, or any of the bone morphogenic proteins (BMPs) BMPs 1-15, any of heregluin/neuregulin/ARIA<y>neodifferentiation factor (ND F) the family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurturin, agrin, et any of the family of semaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggjn, sonic hedgehog and tyrosine hydroxylase.

Other useful transgenic products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), inter-leukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL -12, and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor a and -P, interferons a, p and y, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also usable according to the invention. These include, without limitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T-cell receptors, chimeric T-cell receptors, single-chain T-cell receptors, class I and class IIMHC molecules, as well as engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), degradation accelerating factor (DAF), CR1, CF2 and CD59.

Still other useful gene products include any of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. The invention includes receptors for cholesterol regulation including the low-density lipoprotein (LDL) receptor, high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) receptor and the scavenger receptor. The invention also encompasses gene products as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, vitamin D receptors and other nuclear receptors. In addition, usable gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), aP-1, AP2, myb, Myod and myogenin, ETS box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4 , ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT box binding proteins, interferon regulatory factor (TRF-1), Wilms tumor protein, ETS binding protein, STAT, GATA box binding proteins, such as GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include carbamoyl synthase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetate hydrolase, phenylalanine hydroxylase, α-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VJJI, factor IX, cystathion P synthase, branched-chain keto acid decarboxylase , albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mustase, glutaryl CoA dehydrogenase, insulin, P-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, a cystic fibrosis transmembrane regulator (CFTR ) sequence and a dystrophin cDNA sequence. Still other useful gene products include enzymes such as those that may be useful in enzyme replacement therapy useful in a number of conditions resulting from defective enzyme activity. For example, enzymes containing mannose-6-phosphate can be used in therapies for lysosomal storage diseases (eg, a suitable gene including that encoding P-glucoronidase (GUSB)).

Other useful gene products include non-naturally occurring polypeptides such as chimeric or hybrid polypeptides with a non-naturally occurring amino acid sequence containing insertions, deletions or amino acid substitutions. For example, single-chain engineered immunoglobulins may be useful in certain immunocompromised patients. Other types of non-naturally occurring gene sequences include antisense molecules and catalytic nucleic acids such as ribozymes, which can be used to reduce overexpression of a target.

Reduction and/or modulation of the expression of a gene is particularly desirable for the treatment of hyperproliferative conditions characterized by hyperproliferating cells, such as cancer and psoriasis. Target polypeptides include those polypeptides produced exclusively or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include polypeptides encoded by oncogenes such as myb, mye, fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk, and EGRF. In addition to oncogene products as target antigens, the target polypeptide for anticancer treatment and protective regimens includes variable regions of antibodies made by B-cell lymphomas and variable regions of T-cell receptors of T-cell lymphomas which, in some embodiments, are also used as target antigens for autoimmune disease. Other tumor-associated polypeptides can be used as target polypeptides, for example polypeptides found at higher levels in tumor cells including the polypeptide recognized by monoclonal antibody 17-1A and folate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those that may be useful for treating individuals suffering from autoimmune diseases and disorders by eliciting a broad-based, protective immune response against targets associated with autoimmunity including cell receptors and cells that produce "self"- directed antibodies. T-cell mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (JDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis , Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases is characterized by T-cell receptors (TCRs) that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases.

C. Immunogenic transgenes

Alternatively, or in addition, the vectors according to the invention may contain AAV sequences according to the invention and a transgene which codes for a peptide, polypeptide or protein which induces an immune response against a selected immunogen. For example, the immunogens may be selected from a number of viral families. Examples of desired viral families against which an immune response would be desirable include the picornavirus family which includes genera rhinoviruses, which are responsible for 50% of cases of the common cold; genera enteroviruses that include polioviruses, coxsackieviruses, echoviruses and human enteroviruses such as hepatitis A virus; and genera aptoviruses responsible for foot-and-mouth disease, primarily in non-human animals. Within the picornavirus family of vimser, target antigens include VP1, VP2, VP3, VP4 and VPG. Another viral family includes the calcivirus family comprising the Norwalk group of viruses that are an important causative agent of epidemic gastroenteritis. An additional viral family desirable for bmk in the recognition of antigens to induce immune responses in humans and non-human animals is the togavirus family which includes genera a viruses which include Sindbis vimser, RossRiver vimser and Venezuelan, Eastern and Western equine encephalitis, and mbivims including Rubella vims. The Flaviviridae family includes dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis, and waterborne encephalitis viruses. Other target antigens can be generated from the hepatitis C or coronavirus family which includes a number of non-human VIMs such as infectious bronchitis VIMs (poultry), porcine transmissible gastroenteric VIMs (pigs), porcine hemagglutinating encephalomyelitis VIMs (pigs), feline infectious peritonitis VIMs (cats), feline enteric coronaviruses (cats), canine coronaviruses (dogs), and human respiratory coronaviruses that can cause the common cold and/or non-A, B, or C hepatitis. Within the coronavim family, the target antigens include E1 (also called M or matrix protein), E2 (also called S or Spike protein), E3 (also called HE or hemagglutin-elterosis), glycoprotein (not present in all coronavims), or N (nucleocapsid) . Still other antigens can be seen against the rhabdovirus family which includes genera vesiculoviruses (eg Vesicular Stomatitis viruses) and the general lyssavimes (eg rabies). Within the rhabdovirus family, suitable antigens can be derived from the G protein or the N protein. The family filoviridae which includes haemorrhagic fever viruses such as Marburg and Ebola viruses may be a suitable source of antigens. The paramyxovirus family includes parainfluenza vims type 1, parainfluenza vims type 3, bovine parainfluenza vims type 3, mbulavims (mumpavims, parainfluenza vims type 2, parainfluenza vims type 4, Newcastle disease vims (chickens), rinderpest, morbillivirus, which includes measles and rabbit distemper, and pneumovims which includes respiratory syncytial vims. Influenza virus is classified within the family orotomyxoviurs and is a suitable source of antigen (eg HA protein, NI protein). Bunyavirus family includes genera bunyavims (Californai encephalitis, La Crosose), phlebovirus (Rift Valley Fever) , hantavirus (puremala is a hemahaginous fever vims), nairovims (Nairobi sheep disease) and various unattributed bungavims. Arenavirus family provides a source of antigens against LCM and Lassa fever vims. The reovims family includes the genera reovims, rotavims (causing acute gastroenteritis in children), orbivims and cultiviruses (Colorado Great fever, Lebombo (humans), ek wine encephalosis, blue-tongue).

The retrovirus family includes the subfamily oncorivirinal which includes such human and veterinary diseases as feline leukemia virus, HTLVI and HTLVTI, lentivirinal (which includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine effectual anemia virus and spumavirinal) . Between HIV and SIV, many suitable antigens have been described and can be easily selected. Examples of suitable HIV and SIV antigens include without limitation the gag, pol, Vif, Vpx, VPR, Env, Tat and Rev proteins as well as various fragments thereof. In addition, a number of modifications to these antigens have been described. Suitable antigens for this purpose are well known to those skilled in the art. For example, one can choose a sequence that codes for gag, pol, Vif and Vpr, Env, Tat and Rev, among other proteins, see for example the modified gag protein described in US 5,972,596. See also the HIV and SIV proteins described by D.H. Barouch et al. in "J: Virol.", 75(5):2462-2467 (March 20001), as well as by R.R. Amara et al. in "Science", 292:69-74 (April 6, 2002). These proteins or subunits thereof can be delivered alone, or in combination via separate vectors or from a single vector.

The papovavirus family includes the subfamily polyomaviruses (BKU and JCU viruses) and the subfamily papillomaviruses (associated with cancer and malignant progression of papillomas). The adenovim family includes vims (EX, AD7, ARD, O.B.) which cause respiratory disease and/or enteritis. The parvovim family includes feline parvovims (feline enteritis); feline panleukopenia vims, canine parvovims, and porcine parvovims. The herpesvim family includes the subfamily alphaherpesvirinae, which includes genera sim-plexvims (HSVL HSVIT), varicellovims (pseudorabies, varicella zoster) and the sub-family betaherpesvirinae, which includes the genera cytomegalovirus (HCMV, muromegalovims) and the sub-family gammeherpesvirinae which includes the genera lymphocryptovirus, EBV (Burkitt's lymphoma), infectious rhinotracheitis, Marek's disease vims and rhadinovims. The poxvim family includes the subfamily cordopoxvirinae which includes the genera ortho-poxvims (Variola (smallpox) and vaccinia (cowpox)), parapoxvims, avipoxvims, capripoxvims, leporipoxvims, suipoxvims and the subfamily entomopoxvirinae. The hepadnvavims family includes hepatitis B vimsen An unclassified vims that may be a suitable source for antige ner is Hepatitis delta vimsen. Additional viral sources may include avian infectious bursal disease viruses and porcine respiratory and reproductive syndrome viruses. The alpha vims family includes equine arteritis vims and various Encefa-litis vims.

The present invention may also include immunogens that are useful for immunizing a human or non-human animal against other pathogens including bacteria, fungi, parasitic microorganisms or multicellular parasites that infect human and non-human vertebrates, or form a cancer cell or tumor cell. Examples of bacterial pathogens include pathogenic gram-positive cocci including pneumococci; staphylococci; and streptococci. Pathogenic gram-negative cocci include meningococcus; gonococcus. Pathogenic enteric gram-negative bacilli include enterobacteriaceae; psudomonas, acinetobacteia and eikenella; melioidosis, salmonella; shigella; haemophilus; moraxella; H. Ducreyi (which causes chancroid); bmcella; Franisealla tularensis (which causes tularemia); yersinia (pasteurella); streptobacillus moniliformis and spirillum; Gram-positive bacilli include listeria monocytogenes; erypsipelotrhrix rhusopathye; Corynebacterium diphtheria (diphtheria), klaara; B. Anthracis (anthrax); donovanosis (granuloma inguinale); and bartonellosis. Diseases caused by pathogenic, anaerobic bacteria include tetanus; botulism; other clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic, spirochetal diseases include syphilis; treponematoses; yaws, pinta and endemic syphilis; and leptospirosis. Other infections caused by higher pathogenic bacteria and pathogenic fungi include actinomycosis, nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidiomycosis; candidiasis, aspergillosis, and mucormycosis; sports tricot; paracocci idiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis. Rickettsial infections include typhoid fever, Rocky Mountain spotted fever, Q fever, and Rickettsial-opox. Examples of mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum, psittacosis; and perinatal chlamydial infections. Pathogenic eukaryotes include pathogenic protozoans and helminths and infections from these include: amoebiasis, malaria, leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Tricanes, Toxoplasmo gondii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or "flukes"; and cestosis (tapeworm) infections.

Many of these organisms and/or toxins produced by them have been identified by the Centers for Disease Control [(CDC), Department of Health and Human Services, USA] as having potential for use in biological attacks. For example, some of these biological agents include Bacillus anthracis (anthrax), Clostridium botulinum and its toxin (botulism), Yersinia pestis (plague) variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fever, all in day classified as Category A funds; Coxiella burnetti (Q fever); Brucella species (brucellosis), Burkhol-deria matlei (snive), Ricinus communis and its toxin (ricintoxin), Clostridium per-jringens and its toxin (e-toxin), Stahylococcus species and their toxins (enterotoxin B), all in day classified as Category B funds; and Nipan virus and hantaviruses which are currently classified as Category C agents. In addition, other organisms classified as such or differently may be identified and/or used for such a purpose in the future. It will be readily understood that the viral vectors and other constructs described herein are useful for delivering antigens from these organisms, vims and their toxins or other by-products, which will prevent and/or treat infection or other adverse reactions with these biological agents .

Administration of the vectors according to the invention to deliver immunogens against the variable region of the T cell triggers an immune response including CTLs to eliminate these T cells. In rheumatoid arthritis (RA), various specific, variable areas of T-cell receptors (TCRs) that are involved in the disease have been characterized. These TCRs include V-3, V-14, V-17 and Va-17. Thus, delivery of a nucleic acid sequence encoding at least one of these polypeptides will trigger an immune response that will target T cells involved in RA. In multiple sclerosis (MS), several specific, variable regions of TCRs involved in the disease have been characterized. These TCRs include the V-7 and Va-10. Thus, delivery of a nucleic acid sequence encoding at least one of these polypeptides will elicit an immune response that will target T cells involved in MS. In scleroderma, several specific, variable regions of TCRs have been characterized that are involved in the disease. These TCRs include V-6, V8, V-14 and Va-16, Va-3C, Va-7, Va-14, Va-15, Va-16, Va-28 and Va-12. Thus, delivery of a nucleic acid molecule encoding at least one of these polypeptides will trigger an immune response that will target T cells involved in scleroderma.

Optionally, vectors containing AAV sequences according to the invention can be delivered using a prime-boost regime. A number of such regimes are described in the art and may be readily selected, see for example TO 00/11140, published March 2, 2000, to which reference is made for details.

Such prime-boost regimens typically involve the administration of a DNA (eg, plasmid) based vector to prime the immune system of others, booster, administration with a traditional antigen such as a protein or a recombinant virus carrying the sequences encoding such an antigen. In such an embodiment, the invention provides a method for priming and boosting an immune response against a selected antigen by delivery of a plasmid DNA vector carrying said antigen, followed by boosting, for example with a vector containing AAV sequences according to the invention.

In one embodiment, the prime-boost regimen involves the expression of multiproteins from prime and/or boost carriers, see, for example, R.R. Amara in "Science", 292:69-74 (April 6, 2001) which describes a multiprotein regimen for the expression of protein subunits that can be used to generate an immune response against HIV and SIV. For example, a DNA prime can deliver Gag, Pol, Vif, VPX, and Vpr and Env, Tat, and Rev from a single transcript. Alternatively, SIV Gag, Pol and HIV-1 Env are released.

However, the prime-boost regimens are not limited to immunization for HIV or to the delivery of these antigens. For example, priming may involve delivery with a first chimp vector according to the invention followed by boosting with a second chimp vector, or with a preparation containing the antigen per se in protein form. In one example, the prime-boost regimen may provide a protective immune response against the virus, bacteria, or other organism from which the antigen is delivered. In another desired embodiment, the prime-boost regimen provides a therapeutic effect that can be measured using conventional assays for detecting the presence of the condition for which the therapy is administered.

The priming vaccine can be administered at different sites in the body in a dose-dependent mode that depends on the antigen against which the desired immune response is elicited. The invention is not limited to the amount of injection sites or to the pharmaceutical carrier. Rather, priming steps include treatment regimens that include a single dose or dosage that is administered hourly, daily, weekly, monthly, or annually. As an example, mammals may receive one or two priming injections containing between 10 µg and 50 µg of plasmid in carrier. A desired priming amount or dosage for priming DNA vaccine preparations is between around 1 ug and 10,000 ug of the DNA vaccine. The dosage rings can vary from 1 ug to 1,000 ug DNA per kg individual body weight. The amount or injection site is preferably selected based on the identity and condition of the mammal being treated.

The dosage unit for the DNA vaccine, which is suitable for delivering the antigen to the mammal, is described here. The DNA vaccine is prepared for administration by suspension or solution in a pharmaceutical or physiologically acceptable carrier such as isotonic salt solution, isotonic salts in solution or other formulations that will be obvious to the person skilled in the art. The suitable carrier will be obvious to the person skilled in the art and will depend largely on the route of administration. The preparations according to the invention can be administered to a mammal according to the routes described above in the form of a formulation with sustained release using a biodegradable biocompatible polymer, or by delivery on the seat using micelles, gels and liposomes.

Optionally, the priming step according to the invention can also include, together with the administration of the priming DNA vaccine preparation, a suitable amount of an adjuvant as defined here.

Preferably, a boosting preparation is administered about 2 to about 27 weeks after administration of the priming DNA vaccine to the mammalian subject. The administration of the boosting preparation is carried out using an effective amount of a boosting vaccine preparation containing or able to deliver the same antigen as administered by the priming DNA vaccine. The boosting preparation may consist of a recombinant viral vector derived from the same viral source or from another source. Alternatively, the "boosting preparation" can be a preparation containing the same antigen as encoded in the priming DNA vaccine but in the form of a protein or peptide, which preparation induces an immune response in the host. In another embodiment, the boosting vaccine preparation includes a preparation containing a DNA sequence that encodes the antigen under the control of a regulatory sequence that directs its expression in a mammalian cell, for example vectors such as well-known bacterial or viral vectors. The primary requirements for the boosting vaccine preparation are that the antigen of the vaccine preparation is the same antigen, or a cross-reactive antigen, as encoded by the DNA vaccine.

Preferably, the vectors according to the invention are also suitable for use in regimens that use non-AAV vectors as well as proteins, peptides and/or other biologically usable therapeutic or immunogenic compounds. These regimens are particularly suitable for gene delivery for therapeutic purposes and for immunization, including the induction of protective immunity. Such applications will be readily apparent to those skilled in the art.

Furthermore, a vector according to the invention provides an efficient gene transfer transporter that can deliver a selected transgene to a selected host cell in vivo or ex vivo also where the organism has neutralizing antibodies against one or more AAV serotypes. In one embodiment, the vector (eg, an rAAV) and the cells are mixed ex vivo; the infected cells are cultured using conventional methods; and the transduced cells are reinfused into the patient. Furthermore, the vectors according to the invention can also be used for the production of a desired gene product in vitro. For in vitro production, a desired product (for example a protein) can be obtained from a desired culture after transfection of host cells with an rAAV containing the molecule encoding the desired product and cultivation of the cell culture under conditions that allow expression. The expressed product can then be purified and isolated as desired. Suitable techniques for transfection, cell cultivation, purification and isolation are well known to those skilled in the art.

The following examples shall illustrate various aspects and embodiments of the invention.

EXAMPLES

Example 1:

PCR amplification, cloning and characterization of new AAV sequences.

Tissues from non-human primates are scanned for AAV sequences using a PCR method based on oligonucleotides to highly conserved regions of known AAVs. A stretch of AAV sequence spanning 2886 to 3143 bp of AAV1 [SEQ ID no. 6] was chosen as a PCR amplicon in which a hypervariable region of the capsid protein (Cap) which is unique to each known AAV serotype, and which is here indicated as a "signature region", is flanked by conserved sequences. In later analyses, this signature region was shown to be located between conserved residues spanning hypervariable region 3.

An initial examination of peripheral blood from a number of non-human primate species revealed detectable AAV in a subset of animals from species such as rhesus macaques, cynomolgus macaques, chimpanzees and baboons. However, there was no AAV sequence detected in any of the other tested species including Japanese macaques, pig-tailed macaques and squirrel monkeys. A more extensive analysis of vector distribution was conducted in tissue from rhesus monkeys at the University of Pennsylvania and Tulane colonies recovered at necropsy. This revealed AAV sequence through a wide pattern of tissue.

A. Amplification of an AA V signature area

DNA sequences of AAV1-6 and AAVs isolated from geese and ducks were aligned next to each other using "Clustal W" at default settings. The arrangement for AAV1-6 and inclusion of information for the new AAV7 is given in Figure 1. Sequence similarities among the AAVs were compared.

In this study, a 257 bp region spanning 2886 bp to 3143 bp of AAV1 [SEQ ID No. 6], and the corresponding region in the genomes of the AAV 2-6 genomes [see Figure 1] were identified by the inventors. This area is located at the AAV capsid gene and has highly conserved sequences at both the 5- and 3' ends and has a relatively variable sequence in the middle. In addition, this region contains a DralU restriction enzyme site (CACCACGTC, SEQ ID No. 15). The inventors have found that this region serves as a specific signature for each known type of AAV DNA. In other words, by following PCR reactions, digestion with endonucleases that are specific for each known serotype, and gel electrophoresis analysis, this area can be used to definitively identify amplified DNA as being from serotypes 1, 2, 3, 4, 5, 6, or another serotype.

The primers were designed, validated and the PCR conditions optimized with AAV1, 2 and 5 DNA controls. The primers were based on the sequences of AAV2: the 5' primer, IS: bp 2867-2891 of AAV2 (SEQ ID No. 7) and the 3' primer, 18as, bp 3095-3121 of AAV2 (SEQ ID No. 7).

Cellular DNAs from various tissues including blood, brain, liver, lung, testis and so on from various rhesus monkeys were studied using the strategy described above. The results showed that DNAs from different tissues of these monkeys gave rise to strong PCR amplifications. Further restriction analyzes of the PCR products indicated that they were amplified from AAV sequences that differed from all published AAV sequences.

PCR products (with a size of around 255 bp) from DNAs from a variety of monkey tissue have been cloned and sequenced. Bioinformatics studies on these new AAV sequences suggested that they are new AAV sequences of the capsid gene and distinct from each other. Figure 1 includes the arrangement of the new AAV signature regions for AAV 10-12 [SEQ ID no. 117, 118 and 119 respectively]. Multiple sequence alignment analysis was performed using the Clustal W (1.81) program. The percentage of sequence identity between the signature regions of the AAV 1-7 and AAV 10-12 genomes is given below.

Over 300 clones containing novel AAV serotype sequences spanning the selected 257 bp region were isolated and sequenced. Bioinformatics analysis of these 300+ clones suggested that this 257 bp region is critical for serving as a good land marker or signature sequence for rapid isolation and identification of a new AAV serotype.

B. Use of signature region for PCR amplification

The 257 bp signature region was used as a PCR anchor to extend the PCR amplifications to the 5' of the genome to cover the splice region for the rep and cap genes (1398 bp - 3143 bp, SEQ ID no. 6) and 3' of the genome for to obtain the entire cap gene sequence (2866 bp - 4600 bp, SEQ ID no. 6). PCR amplifications were carried out using the standard conditions including denaturation at 95 °C for 0.5-1 minute, annealing at 60-65 °C for 0.5-1 minute and extension at 72 °C for 1 minute per kb with a total number of amplification cycles from 28 to 42.

Using the aligned sequences as described under point "A", two other relatively conserved regions were identified in the sequence located at the 3' end of rep genes and 5' to the 257 bp region and in the sequence downstream of the 57 bp fragment but before the AAV 3 ITR. Two sets of new primers were designed and the PCR conditions optimized for recovery of total capsid or part of the rep sequences of new AAV serotypes. More specifically, for the 5' amplification, the 5' primer was AVINs, GCTGCGTCAACTGGACCAATGAGAAC [nt 1398-1423 of AVI, SEQ ID No. 6] and the 3' primer was 18as, identified above. For 3' amplification, the 5' primer was ls, identified above, and the 3' primer was Av2Las, TCGTTTCAGTTGAACTTTGGTCTCTGCG [nt 4435-4462 of AAV2, SEQ ID No. 7].

In these PCR amplifications, the 257 bp region was used as a PCR anchor and land marker to generate overlapping fragments to construct a complete capsid gene

by fusion at the DRAin site in the signature region followed by amplification of the 5- and 3' extension fragments, obtained as described herein. More specifically, to generate the intact AAV 7 cap gene, the three amplification products were (a) the sequences of the signature region; (b) the sequences of the 5' extension; and (c) the sequences of the 3' extension, cloned into a

pCR4-Topo [Invitrogen] plasmid backbone according to the manufacturer's instructions. The plasmids were then digested with Drain and recombined to form an intact cap gene.

In this way of working, around 80% of the capsid sequences of AAV7 and AAV8 were isolated and analyzed. Another new serotype, AAV9, was also detected from monkey #2.

Using the PCR conditions as described above, the remaining part of the rep gene sequence for AAV7 is isolated and then cloned using primers that amplify 108 bp to 1461 bp of the AAV genome (calculated using the numbering of AAV2, SEQ ID no. 7) . This clone is sequenced to construct a complete AAV7 genome without ITRs.

C. Direct amplification of the 3.1 kb Cap fragment

To directly amplify a 3.1 kb full-length Cap fragment from NHP tissue and blood DNAs, two other highly conserved regions were identified in AAV genomes for use in PCR amplification of large fragments. A primer in a conserved region located in the middle of the rep gene was chosen (AVlns: 5' GCTGCGTCAACTGGACCAATGAGAAC 3', nt 1398-1423 of SEQ ID no. 6) in combination with the 3' primer located in another conserved region downstream of the cap gene ( AV2cas: 5' CGCAGAGACCAAAGTTCAACTGAAACGA 3', SEQ ID No. 7) for amplification of full-length cap fragments. The PCR products were Topo-cloned according to the manufacturer's guidelines (Invitrogen) and the sequence analysis was performed with Qiagengenomics (Qiagengenomics, Seattle, WA) with an accuracy of >99.9%. A total of 50 capsid clones were isolated and characterized. Among these were 37 clones derived from Rhesus macaque tissue (rh. 1 - rh.37), 6 clones from cynomologous macaques (cy. 1 - cy.6), 2 clones from baboons (bb.l and bb.2) and 5 clones from chimpanzees (ch.l - ch.5).

To rule out the possibility that the sequence diversity in the new AAV family was not a result of PCR, such as PCR-mediated gene splicing by overlap extension between different partial DNA templates with homologous sequences, or the result of recombination processes in bacteria, a series of experiments was carried out under identical conditions for VP1 amplification using total cellular DNAs. First, intact AAV7 and AAV8 plasmids were mixed in an equal molar ratio followed by serial dilutions. The serially diluted mixtures were used as templates for PCR amplification of 3.1 kb VP1 fragments using universal primers and identical PCR conditions to that used for DNA amplification to see whether any hybrid PCR products were generated. The mixture was transformed into bacteria and isolated transformants to look for hybrid clones that could possibly arise from recombination processes in bacterial cells. In another experiment, AAV7 and AAV8 plasmids were restriction treated with Msp I, Ava I and Hael, where all cut both genomes several times at different positions, then mixed the digests in different recombinations and used these for PCR amplification of VP1 fragments under the same conditions to test whether PCR products could be generated by overlap sequence extension of partial AAV sequences. In another experiment, a mixture of gel-purified 5' 1.5 kb AAV7 VP1 fragment and 3' 1.7 kb AAV8 VP1 fragment with overlap in the signature region was serially diluted and used for PCR amplification in the presence and absence of 200 ng of cellular DNA, extracted from a monkey cell line that was free of AAV sequences by TaqMan analysis. None of these experiments showed efficient PCR mediated overlap sequence production under the condition of the genomic DNA cap amplification (data not shown). As a further confirmation, 3 pairs of primers that were located in different HVRs and were sequence specific to the variants of clone 42s from Rhesus macaque F953, in different combinations, were designed to amplify shorter fragments of mesenteric lymph node (MLN) DNA from F953 from which clone 42s was isolated. All sequence variations identified in full-length cap clones were found in these short fragments (data not shown).

Example 2: Adeno-associated vomers undergo substantial development in primates during natural infections.

Sequence analysis of selected AAV isolates showed divergence through the genome which is the most

concentrated in hypervariable regions of the capsid proteins. Epidemiological data suggest that all known serotypes are endemic to primates, although isolation of clinical isolates is limited to AAV2 and AAV3 from anal and laryngeal scrapings of human children and AAV5 from a human condylomatous wart. No known clinical sequelae are associated with AAV infection.

In an attempt to better understand the biology of AAV, non-human primates were used as models to characterize the sequelae of natural infections. Tissues from non-human primates were searched for AAV sequences by bmk of the PCR method according to the invention based on the oligonucleotide to highly conserved regions of known AAVs (see example 1). A stretch of AAV sequence over 2886 to 3143 bp of AAV1 [SEQ ID No. 6] was chosen as a PCR amplicon in which conserved sequences are flanked by a hypervariable region that is unique to each known AAV serotype, here designated as a "signature region" .

An initial examination of peripheral blood from a number of non-human primate species including rhesus monkeys, cynomolgus monkeys, chimpanzees and baboons showed detectable AAV in a subset of animals from all species. A more extensive analysis of vector distribution was conducted in tissue from rhesus monkeys from the University of Pennsylvania and Tulane colonies, recovered at necropsy. This showed ata AAV sequences throughout a wide pattern of tissues.

The amplified signature sequence was subcloned into plasmids and individual transformants were subjected to sequence analysis. This showed substantial variation in the nucleotide sequence of clones derived from different animals. Variation in the signature sequence was also noted in clones obtained within individual animals. Tissues obtained from two animals in which unique signature sequences were identified (ie, colon from 98E044 and heart from 98E056) were further characterized by extending the sequence amplified by PCR using oligonucleotides to highly conserved sequences. In this way, complete proviral structures were reconstructed for viral genomes from both tissues as described here. These proviruses differ from the known AAVs with the greatest sequence divergence noted in the regions of the Cap gene.

Further experiments were conducted to confirm that the AAV sequences resistant to non-human private tissue represented proviral genomes of infectious viruses capable of being retrieved and forming virions. Genomic DNA from liver tissue of animal 98E056 from which the AAV8 signature sequence was determined was digested with an endonuclease that does not have a site within the AAV sequence and transfected into 293 cells with a plasmid containing an E1 deleted genome of human adenovirus serotype 5 as a source of helper functions . The resulting lysate was applied to 293 cells once and the lysate recovered and analyzed for the presence of AAV cap proteins by bmk of a broadly reactive, polyclonal antibody against cap proteins and the presence and abundance of DNA sequences from the PCR amplified AAV provims from which AAV8 was derived . Transfection of endonuclease restriction-treated heart DNA and the adenovims helper plasmid yielded high amounts of AAV8 vims as demonstrated by detection of cap proteins by Western blot analysis and the presence of IO<4>AAV8 vector genomes per 293 cells. Lysates were generated from a large-scale preparation and AAV was purified by cesium sedimentation. The purified preparation showed 26 nm icosahedral structure that appeared identical to that of AAV serotype 2. Transfection with the adenovirus helper alone did not yield AAV proteins or genomes, ruling out contamination as a source of recovered AAV.

To further characterize the inter- and intra-animal variation of the AAV signature sequence, selected tissues were subjected to unextended PCR to amplify the entire Cap open reading frames.

The resulting fragments were cloned into bacterial plasmids and individual transformants were isolated and sequenced completely. This analysis involved mesenteric lymph nodes from three rhesus monkeys (Tulane/V223 - 6 clones; tulane/T612 - 7 clones; Tula-ne/F953 - 14 clones), livers from two rhesus monkeys (Tulane/V251 - 3 clones; Penn/00E033 - 3 clones), spleen from a rhesus monkey (Penn/97E043 - 3 clones), heart from a rhesus monkey (JHGT/98E046- 1 clone) and peripheral blood from a chimpanzee (New Iberia/X133 - 5 clones), six cynomolgus macaques (Charles River/A1378, A3099, A3388, A3442, A2821, A3442 - a total of 6 clones) and a baboon (SFRB/8644 - 2 clones). Of the 50 clones sequenced from 15 different animals, 30 were considered non-abundant based on the finding of at least 7 amino acid differences from each other. The non-redundant VP1 clones were numbered sequentially as they were isolated with a prefix indicating the species of non-human primate from which they were obtained. The structural relationship between these 30 non-redundant clones and the previously described 8 AAV serotypes was determined using the SplitsTree program [D.H. Huson in "SplitsTree: analyzing and visualizing evolutionary data." "Bioinformatics" 14, 68-73 (1998)] with implementation of the split decomposition method. The analysis indicates homoplasy between a set of frequencies in a tree-like network rather than a tree that splits in two. The advantage is to enable the detection of groupings that are the result of convergence and to show phylogenetic relationships even when they are disturbed by parallel events. Extensive phylo-genetic research will be necessary to elucidate AAV development, the intention here is only to group different clones in terms of their sequence similarity.

To confirm that the new VP1 sequences were derived from infectious viral genomes, cellular DNA from tissues with a high abundance of viral DNA was treated with an endonuclease that would not cleave in AAV and then transfected into 293 cells, followed by infection with adenovirus . This resulted in "rescue" and amplification of AAV genomes from DNA from tissues from two different animals (data not shown).

VP1 sequences of the new AAVs were further characterized with regard to the nature and location of the amino sequence variation. All 30 VP1 clones that were shown to differ from each other by more than 1% amino acid sequence were aligned and scored for variation in each residue. An algorithm developed to determine areas of sequence divergence yielded 12 hypervariable regions (HVR) of which 5 overlapped or are part of the 4 previously described variable regions [Kotin, supra; Rutledge, supra]. The three-fold proximal peaks contain most of the variability (HVR5-10). Interestingly, the loops that were located at the 2- and 5-fold axis also showed intense variation. The HVRs 1 and 2 occur in the N-terminal part of the capsid protein which is not resolved in the X-ray structure and suggests that the N-terminus of the VP1 protein is exposed on the surface of the virion.

Real-time PCR was used to quantify AAV sequences from tissues of 21 rhesus monkeys using primers and probes on highly conserved regions of Rep (one set) and Cap (two sets) of known AAVs. Each data point represents analysis of tissue DNA from an individual animal. This confirmed the wide distribution of AAV sequences, even if the quantitative distribution was different between the animals. The source of animal and previous history or treatment did not appear to affect the distribution of AAV sequences in rhesus macaques. Three different sets of primers and probes were used to quantify AAV and gave consistent results. The highest levels of AAV were found consistently in mesenteric lymph nodes at a mean of 0.01 copies per diploid genome for 13 animals that were positive. Liver and spleen also contained high abundance of viral DNA. There were examples of very high AAV such as in the heart of the rhesus macaque 98E056, the spleen of the rhesus macaque 97E043 and the liver of the rhesus macaque RQ4407, which showed 1.5, 3 and 20 copies of the AAV sequence per diploid genome respectively. Relatively high levels of viral DNA were noted in peripheral blood mononuclear cells, suggesting that tissue data are not due to resident blood components (data not shown). It should be pointed out that this method will not necessarily capture all AAVs that are resident in the non-human primates because the detection requires high homology both with regard to the oligonucleotides and the real-time PCR probe. Tissues from animals with a high abundance of AAV DNA were further analyzed for the molecular state of the DNA in question by DNA hybridization techniques, and its cellular distribution, by in situ hybridization.

The type of sequence variation uncovered in AAV proviral fragments isolated from different animals and within tissues from the same animal is reminiscent of the evolution that occurs for many RNA viruses during a pandemic or even within the infection of an individual. In some situations, the notion of a wild-type vims is replaced by the existence of swarms of quasispecies that evolve as a result of rapid replication and mutations in the presence of selective pressure. One example is infection with HIV, which develops in response to immunological and pharmacological pressure. Several mechanisms contribute to the high rate of mutations in RNA vims including low fidelity and lack of reliable readout capacity by reverse transcriptase and non-homologous and homologous recombination.

Evidence for the formation of quasispecies of AAV was illustrated in this study by the systematic sequencing of multiple cloned proviral fragments. Thus, identical sequences could not be found within any extended clones isolated between or within animals. An important mechanism for this evolution of sequence appears to be the high degree of homologous recombination between a more limited number of parenteral genes. The net result is extensive switching of hypervariable regions of the cap protein leading to a pattern of chimeras that may have different tropisms and serological specificities (eg, the ability to escape immunological responses, especially when neutralizing antibodies are involved). Mechanisms by which homologous recombination can occur are unclear. One possibility is that the + - and - strands of different single-stranded AAV genomes anneal during replication as described under high multiplicity of infections with AAV recombinants. It is unclear whether other mechanisms contribute to sequence development in AAV infections. The overall rate of mutation that occurs during AAV replication appears to be relatively low and the data do not suggest high frequencies of replication errors. However, substantial rearrangements of the AAV genome have been described during lytic infection leading to the formation of defective, interfering particles. Regardless of the mechanisms leading to sequence divergence, with few exceptions, the vpl structure of the quasispecies remained intact without frameshift or non-sense mutation, suggesting that competitive selection of vims with the most favorable fitness profile contributes to population dynamics.

These studies have implications in several areas of biology and medicine. The concept of

rapid vims development, previously thought to be a property limited to RNA vims, should be taken into account in DNA vims that have classically been characterized by serological analyses. It will be important expressed in parvoviruses to develop a new method to describe vim isolates that captures the complexity of their structure and biology as in HIV, which are categorized as general families of similar structure and function called Clades. An alternative strategy is to continue to categorize isolates for serological specificity and to develop criteria to describe variants within serological groups.

Example 3: Vectorology for recombinant AAV genomes equipped with AAV2 ITRs by bmk of chimeric plasmids containing AAV2 rep and new AAV cap genes for serological and gene transfer studies in different animal models.

Chimeric packaging constructs are generated by fusing AAV2 rep with cap sequences of new AAV serotypes. These chimeric packaging constructs are initially used for pseudotyping of recombinant AAV genomes carrying AAV2 ITRs by triple transfection in 293 cells using the Ad5 helper plasmid. These pseudotyped vectors are used to evaluate the performance in transduction-based, serological studies and evaluate the gene transfer efficiency of new AAV serotypes in different animal models including NHP and rodents, before isolating intact and infectious vims of these new serotypes.

A. pAAV2GFP

The AAV2 plasmid containing the AAV2 ITRs and green fluorescent protein expressed under the control of a constitutive promoter. This plasmid contains the following elements: the AAV2 ITRs, a CMV promoter, and the GFP coding sequence.

B. Cloning of trans plasmid

To construct the chimeric transplasmid for production of recombinant, pseudotyped AAV7 vectors, the p5E18 plasmid (Xiao et al., 1999, "J. Virol." 73:3994-4003) was partially digested with Xho I to linearize the plasmid by Xho I the seat only in position 3169. The Xho I cut ends were then filled in and ligated back. This modified p5E18 plasmid was treated with Xba I and Xho I in a complete digestion to remove the AAV2 cap gene sequence and replaced with a 2267 bp Spe I/Xho fragment containing the AAV7 cap gene that was isolated from pCRAAV7 6-5+15-4 plasmid.

The resulting plasmid contains the AAV2 rep sequences for Rep78/68 under the control of the AV2 P5 promoter and the AAV2 rep sequences for Rep52/40 under the control of the AAV2 P19 promoter. The AAV7 capsid sequences are under the control of the AAV2 P40 promoter, this plasmid also contains a spacer 5' of the rep ORF.

C. Preparation of pseudotyped rAA V

The rAAV particles (AAV2 vector in AAV7 capsid) are generated using an adenovim-free method. Briefly, the cis plasmid (pAAV2.1 lacZ plasmid containing the AAV2 ITRs) and the trans plasmid pCRAAV7 6-5+15-4 (containing AAV2 rep and AAV7 cap) and a helper plasmid, respectively, were simultaneously cotransfected into 293 cells in a ratio of 1:1:2 by calcium phosphate precipitation.

For construction of the pAd helper plasmids, the pBG10 plasmid was purchased from Microbix (Canada). An RsII fragment containing L2 and L13 was deleted from pBHG10, yielding the first helper plasmid, pAdAF13. Plasmid AdA F1 was constructed by cloning the Asp700/Sall fragment with a Pme1/Sgf1 deletion, isolation from pBHG10 inside Bluescript. MLPL, L2 and L3, were deleted in pAdAF1. Further deletions of a 2.3 kb Nrul fragment and then a 0.5 kb RsrH/NruI fragment generated helper plasmids pADAF5 and pADAF6, respectively. The helper plasmid, named pAF6, provided the essential helper functions of E2a and E4 ORF6 that were not provided by the E1-expressing Hel parcel, but are deleted from adenoviral capsid proteins and functional E1 regions.

Typically, 50 µg DNA (cis:trans:helper) was transfected onto a 150 mm tissue culture dish. The 293 cells were harvested 72 h post-transfection, sonicated and treated with 0.5% sodium deoxycholate (37°C for 10 min). Cell lysates were then subjected to two rounds of a CsCl gradient. Peak fractions containing rAAV vector were collected, pooled and dialyzed against PBS.

Example 4: Formation of infectious clones carrying intact new AAV serotypes for study of principle virology in human and NHP derived cell lines and assessment of pathogenesis of new AAV serotypes in NHP and other animal models.

To achieve this aim, the genome walker system was used to obtain 5'- and 3- terminal sequences (ITRs) and complete construction of clones containing intact, new AAV serotype genomes.

Briefly, using a commercially available "Universal Genome Walker Kit" [Clontech], genomic DNAs from monkey tissues or cell lines identified as positive for the presence of the AAV7 sequence were digested with Dra I-, EcoR V-, Pvu II- and Stu I endonuclease and ligated to the "Genome Walker Adaptor" to generate 4 individual "Genome Walker Libraries" (GWLs). Using DNAs from the GWLs as templates, AAV7 and neighboring genomic sequences are PCR-amplified by adapter primer 1 (API, provided in the kit) and an AAV7-specific primer 1, followed by a nested PCR using adapter primer 2 ( AP2) and an additional AAV7 specific primer 2, both internal to the first set of primers. The main PCR products from the "nested" PCR are cloned and characterized by sequence analysis.

In this experiment, primers covering the 257 bp or other signature fragments of a generic AAV genome were used for PCR amplification of cellular DNAs extracted from Human- and NHP-derived cell lines to identify and characterize the latent AAV sequences. The identified, latent AAV genomes are recovered from the positive cell lines using adenovirus helpers of different species and strains.

To isolate infectious AAV clones from NHP-derived cell lines, a desired cell line is obtained from ATCC and screened by PCR to identify the 257 bp amplicon, that is the signature region of the invention. This 257 bp PCR product is cloned and serotyped by sequencing analysis. For these cell lines containing the AAV7 sequence, the cells are infected with SV-15, a simian adenovirus acquired from ATCC, human Ad5 or transfected with a plasmid construct harboring the human Ad genes responsible for the AAV helper functions. 48 hours after infection or transfection, cells are harvested and Hirt DNA prepared for cloning of AAV7 genome according to Xiao et al., 1999, "J. Virol.", 73:3994-4003.

Example 5: Production of AAV vectors

A pseudotyping strategy similar to that in example 3 for AAV1/7 was used to

produce AAV2 vectors packaged with AAV1, AAV5 and AAV8 capsid proteins. Briefly, recombinant AAV genomes equipped with AAV2 ITRs are packaged by triple transfection of 293 cells with cis-plasmid, adenovirus helper plasmid and a chimeric packaging construct in which the AAV2 rep gene is fused with cap genes of new AAV serotypes. To create the chimeric packaging constructs, the Xho I site of the p5E18 plasmid was ablated at 3169 bp and the modified plasmid was treated with Xba I and Xho I in a complete digest to remove the AAV2 cap gene and replace it with a 2267 bp Spe I/Xho In fragment containing the AAV8 cap gene [Xiao, W., et al., (1999) "J Virol" 73, 3994-4003]. A similar cloning strategy was used to create chimeric packaging plasmids of AAV2/1 and AAV2/5. All recombinant vectors were purified by the standard CsCb sedimentation method except AAV2/2 which was purified by a single step heparin chromatography.

Genome copy (GC) titers of AAV vectors were determined by TaqMan assay using probes and primers targeting the SV40 poly A region as described previously [Gao, G, et al., (2000) "Hum Gene Ther" 11, 2079- 91].

Vectors were constructed for each serotype for a number of in vitro and in vivo studies. Eight different transgenic cassettes were incorporated into the vectors and recombinant virions were prepared for each serotype. The recovery of virus, based on genome copy number, is summarized in Table 4. Yields of vector were high for each serotype with no consistent differences between serotypes. Data shown in the table are mean genome copy yields with standard deviation x 10<13> of multiple production lots of 50 plate (150 mm) transfections.

Example 6: Serological analyzes of pseudotyped vectors

C57BL/6 mice were injected with vectors of different serotypes of AAVCBA1AT vectors intramuscularly (5 x 10<11> ) and serum samples were collected 34 days later. To test the neutralizing and cross-neutralizing activity of sera against each serotype of AAV, sera were analyzed in a transduction-based neutralizing antibody assay [Gao, G.P., et al., (1996) "J Virol", 70, 8934-43]. More specifically, the presence of neutralizing antibodies was determined by assessing the ability of serum to inhibit transduction of 84-31 cells with reporter viruses (AAVCMVEGFP) of different serotypes. Specifically, the reporter virus AAVCMVEGFP of each serotype [at multiplicity of infection (MOI) leading to transduction of 90% of the indicator cells] was preincubated with heat-inactivated serum from animals that had received different serotypes of AAV from naïve mice. After 1 hour of inhibition at 37 °C, the viruses were added to the 84-31 cells in 96-well plates for 48 or 72 hours, depending on the virus serotype. The expression of GFP was measured by Fluor-olmagjn (Molecular Dynamics) and quantified by "Image Quant Software". Neutralizing antibody titers were reported as the highest serum dilution that inhibited transduction to less than 50%.

The availability of GFP-expressing vectors facilitated the development of an assay for neutralizing antibodies based on inhibition of transduction in a permissive cell line (that is, 293 cells stably expressing E4 from Ad5). Sera to selected AAV serotypes were generated by intramuscular injection of the recombinant vims. Neutralization of the AAV transduction by 1:20 and 1:80 dilutions of antisera was evaluated (see Table 5). Antisera to AAV1, AAV2, AAV5 and AAV8 neutralized transduction of the serotypes to which the antiserum was generated (AAV5 and AAV8 to a lesser extent than AAV1 and AAV2) but not to the other serotype (that is, there was no evidence of any cross-neutralization suggesting that AAV8 is a truly unique serotype).

Human sera from 52 normal individuals were screened for neutralization against selected serotypes. No serum sample was found to neutralize AAV2/7 and AAV2/8 while AAV2/2 and AAV2/1 vectors were neutralized in 20% and 10% sem, respectively. A fraction of human pooled IgG representing a pool of 60,000 individual samples did not neutralize AAV2/7 and AAV2/8, while the AAV2/2 and AAV2/1 vectors were neutralized at titers equal to 1:1280 and 1: 640.

Example 7: In vivo evaluation of different serotypes of AAV vectors

In this study, 7 recombinant AAV genomes, AAV2CBhAl AT, AAV2AlbhA1 AT, AAV2CMVrhCG, AAV2TBGrhCG, AAV2TBGcFIX, AAV2CMVLacZ and AAV2TBGLacZ were packaged with capsid proteins of different serotypes. In all seven constructs, the minigene cassette was flanked by AAV2 ITRs. cDNAs of human α-antitrypsin (AlAT) [Xiao, W., et al., (1999) "J Virol" 73, 3994-4003], the P subunit of rhesus monkey choriogonadotropic hormone (CG) [Zoltick, P.W. & Wilson, J.M. (2000) "Mol Ther" 2,657-9], canine factor IX- [Wang, L., et al., (1997) "Proe Nati Acad Sei USA" 94,11563-6] and bacterial β-galactosidase (it will si Lac Z) genes were used as reporter genes. For liver-directed gene transfer, either the mouse albumin gene promoter (Alb) [Xiao, W. (1999), supra] or the human thyroid hormone binding globulin gene promoter (TBG) [Wang (1977), supra] was used to drive liver-specific expression of rapport genes. In muscle-directed gene transfer experiments, either the cytomegalovirus early promoter (CMV) or the chicken β-actin promoter with the CMV enhancer (CB) was used to drive the expression of reporters.

For muscle-directed gene transfer, vectors were injected into the right tibialis anterior of 4-6 week old nude NCR or C57BL/6 mice (Taconic, Germantown, NY). In liver-directed gene transfer studies, vectors were infused intraportally into 7-9 week old nude NCR or C57BL/6 mice (Taconic, Germantown, NY). Serum samples were collected intraorbitally at various time points after the vector administration. Muscle and/or liver tissue was harvested at various time points for cryosectioning and Xgal histochemical staining from animals receiving the lacZ vectors. For the re-administration experiment, C56BL/6 mice first received AAV2/1-, -2/2-, -2/5-, -2/7-, and -2/8CBA1 AT vectors intramuscularly and followed for Al AT gene expression for 7 weeks. The animals were then treated with AAV"(8TBGcFDC intraportally and studied for cFJX gene expression.

ELISA based assays were performed to quantify the serum levels of hAl AT, rhCG and cFIX proteins as described previously [Gao, G.P., et al, (1996) "J Virol" 70, 8934-43; Zoltick, P.W. & Wilson, J.M. (2000) "Mol Ther" 2, 657-9; Wang, L., et al., "Proe Nati Acad Sei USA" 94, 11563-6]. The experiments were completed when the animals were sacrificed for harvesting of muscle and liver tissue for DNA extraction and quantitative analysis of genome copies of vectors present in the target tissue by TaqMan using the same set of primers and probes as when titrating the vector preparations [Zhang, Y. , et al., (2001) "Mol Ther" 3, 697-707].

The performance of vectors based on the new serotypes was assessed in murine models of muscle- and liver-directed gene transfer and compared with vectors based on the known serotypes AAV1, AAV2 and AAV5. Vectors expressing secreted proteins (α-antitrypsin (Al AT) and chorionic gonadotropin (CG)) were used to quantify relative transduction efficiencies between different serotypes by ELISA analysis of sera. The cellular distribution of transduction within the target organ was assessed using lacZ expressing vectors and Z-gal histochemistry.

The performance of AAV vectors in the skeletal muscle was analyzed after direct injection into the tibialis anterior muscles. Vectors contained the same AAV2-based genome with the immediate early gene of CMV or a CMV enhanced β-actin promoter, driving the expression of the transgene. Early studies indicated that immunocompetent C57BL/6 mice elicited limited humoral responses to the human Al AT protein when expressed from AAV vectors [Xiao, W. et al., (1999) "J Virol" 73, 3994-4003].

In each strain, AAV2/1 vectors produced the highest levels of Al AT and the AAV2/2 vector the lowest, while the AAV2/7 and AAV2/8 vectors showed intermediate levels of expression. Peak levels of CG 28 days after the injection of nu/nu NCR mice showed the highest levels from AAV2/7 and the lowest from AAV2/2, while AAV2/8 and AAV2/1 were in between. Injection of AAV2/1 and AAV2/7 lacZ vectors produced gene expression at the injection sites in all muscle fibers with significantly fewer lacZ positive fibers observed with AAV2/2 and AAV2/8 vectors. These data suggest that the transduction efficiency with AAV2/7 vectors in skeletal muscle is similar to that achieved with AAV2/1 which is the most efficient in skeletal muscle of the previously described serotypes [Xiao, W. (1999), cited above, Chao, H., et al., (2001), "Mol Ther" 4, 217-22; Chao, H., et al., (2000) "Mol Ther" 2, 619-23].

Corresponding murine models were used to evaluate liver-directed gene transfer. Identical doses of vectors based on genome copies were infused into the portal veins of mice which were then analyzed for expression of the transgene. Each vector contained an AAV2-based genome using previously described liver-specific promoters (ie, albumin or thyroid hormone binding globulin) to drive expression of the transgene. More specifically, CMVCG and TBGCG minigene cassettes were used for muscle and liver-directed gene transfer. Levels of rhCG were defined as relative units (Rus x IO<3>). Data are given from analysis of serum samples collected on day 28 after vector administration (4 animals per group). As shown in Table 3, the effect of capsid proteins on the efficiency of transduction of Al AT vectors in nu/nu and C57BL/6 mice and CG vectors in C57BL/6 mice was consistent (see Table 6).

In all cases, AAV2/8 vectors gave the highest levels of transgene expression that ranged from 16 to 110 greater than that obtained with AAV2/2 vectors; expression from AAV2/5 and AAV2/7 vectors was intermediate, with AAV2/7 higher than AAV2/5. Analyzes of X-Gal stained liver sections from animals receiving the corresponding lacZ vectors showed a correlation between the number of transduced cells and the total levels of transgene expression. DNAs extracted from the livers of C57BL/6 mice receiving the Al AT vectors were analyzed for the presence of vector DNA using real-time PCR technology.

The amount of vector DNA found in the liver 56 days after injection correlated with the levels of transgene expression (see Table 7). For this experiment, a set of probe and primers targeting the SV40 polyA region of the vector genome was used for TaqMan PCR. The values shown are the mean of three individual animals with standard deviation. The animals were sacrificed on day 56 to harvest liver tissue for DNA extraction. These studies suggest that AAV8 is the most efficient vector for liver-directed gene transfer due to increased numbers of transduced hepatocytes.

The serological data described above suggest that AAV2/8 vectors should not

is neutralized in vivo after immunization with the other serotypes. C57BL/6 mice that received intraportal injections of AAV2/8 vectors express canine factor JX (10<11> genome copies) 56 days after they received intramuscular injections of Al AT vectors from different serotypes. High levels of factor JX expression were achieved 14 days after infusion of AAV2/8 in naïve animals (17±2 ug/ml, n=4), which was not significantly different from that observed in animals immunized with AAV2 /1 (31±23 µg/ml, n=4), AAV2/2 (16 µg/ml, n=2) and AAV2/7 (12 µg/ml, n=2). This is in contrast to what was observed in AAV2/8 immunized animals infused with AAV2/8 factor JX vector where no detectable factor JX was observed (<0.1 µg/ml, n=4).

Oligonucleotides to conserved regions of the cap gene amplified sequences from rhesus monkeys representing unique AAVs. Identical cap signature sequences were found in many tissues from rhesus monkeys originating from at least two different colonies. Full-length rep and cap open reading frames were isolated and sequenced from individual sources. Only the capped open reading frames of the new AAVs were needed to evaluate their potential as vectors because vectors with the AAV7 or AAV8 capsids were generated using the ITRs and rep from AAV2. This also simplified the comparison between different vectors because the relevant vector genome is identical between different vector serotypes. Thus, the yields of recombinant vectors generated using this route were not significantly different between the serotypes.

Vectors based on AAV7 and AAV8 appeared to be immunologically distinct (that is, they were not neutralized by antibodies generated against other serotypes). Furthermore, sera from humans did not neutralize transduction with AAV7 and AAV8 vectors, which is a significant advantage compared to the human-derived AAVs currently under development for which a significant proportion of the human population has pre-existing immunity that is neutralizing [ Chirmule, N., et al., (1999) "Gene Ther" 6, 1574-83].

The tropism of each new vector is favorable for in vivo applications. The AAV2/7 vectors appear to transduce skeletal muscle as efficiently as AAV2/1 which is the serotype that provides the highest level of transduction in skeletal muscle of primate AAVs tested to date [Xioa., W., supra; Chou (2001), supra and Chou (2000) supra]. Importantly, AAV2/8 provides a significant advantage compared to the other serotypes expressed by efficiency of gene transfer to the liver, which until now has been relatively disappointing expressed by the number of hepatocytes that were stably transduced. AAV2/8 consistently achieved a 10- to 100-fold improvement in gene transfer efficiency compared to the other vectors. The basis for the improved efficiency for AAV2/8 is unclear, although it is possibly due to uptake via a different receptor that is more active in the basolateral surface of the hepatocytes. This improved efficiency will be rather useful in the development of liver-directed gene transfer where the number of transduced cells is critical, for example as is the case with urea cycle disorders and hereditary hypercholesterolemia.

Thus, the invention provides a new approach for isolating new AAVs based on PCR recovery of genomic sequences. The amplified sequences were easily incorporated into vectors and tested in animals. The lack of pre-existing immunity to AAV7 and the favorable tropism of the vectors for muscle suggest that AAV7 is suitable for use as a vector in human gene therapy and other in vivo applications. Similarly, the lack of pre-existing immunity against the AAV serotypes according to the invention, and their tropisms, also make them useful for delivery of therapeutic molecules and other useful molecules.

Example 9 - Tissue tropism studies

In the construction of a high-throughput functional screening scheme for new AAV constructs, a non-tissue specific and highly active promoter, the CB promoter (CMV enhanced chicken p actin promoter) was chosen to drive an easily detectable and quantifiable reporter gene, the human α anti-trypsin gene . Thus, one only needs to make a vector for each new AAV clone for the gene transfer study which targets 3 different tissues, liver, lung and muscle, for screening for tissue tropism for a particular AAV construct. Table 8 summarizes data generated from 4 new AAV vectors in the tissue tropism studies (AAVCBA1 AT), from which a new AAV capsid clone, 44.2, was found to be a very potent gene transfer carrier in all 3 tissues with a wide range in particular lung tissue . Table 8 reports data obtained (in ug Al AT/ml serum) on study day 14.

A couple of other experiments were then carried out to confirm the superior tropism for AAV 44.2 in lung tissue. First, the AAV vector-carried CClOhal AT minigene for lung-specific expression pseudotyped with capsids of new AAVs was given to immunodeficient animals (nude NCR) in an equal volume (50 µl each of the original preparations without dilution) via intratracheal injections as given in Table 9.1 Table 9 is shown 50 µl of each original preparation per mouse, NCR nude, with detection limit >0.033 ug/ml, day 28.

The vectors were also administered to immunocompetent animals (C57BL/6) in equal genome copies (1 x 10<11>) as shown in Table 10. (1 x 10<11>GC per animal, C57BL/6, day 14, limit of detection >0.033 ug/ml)

Data from both trials confirmed the superior tropism of clone 44.2 in lung-directed gene transfer.

Interestingly, the performance of clone 44.2 in liver- and muscle-directed gene transfer was also outstanding, close to that of the best liver transducer, AAV8, and the best muscle transducer, AAV1, suggesting that this new AAV has some special biological significance. chance.

To study serological properties of these new AAVs, pseudotyped AAVGFP vectors were created for immunization of rabbits and in vitro transduction of 84-31 cells in the presence and absence of antisera against the different capsids. Data are summarized below:

Example 10 - Mouse model for hereditary hypercholesterolemia

The following experiments show that the AAV2/7 construct according to the invention emits the LDL receptor and expresses the LDL receptor in an amount sufficient to reduce the levels of plasma cholesterol and triglycerides in animal models of hereditary hypercholesterolemia.

A. Vector construction

AAV vectors, packaged with AAV7 or AAV8 capsid proteins were constructed using a pseudotyping strategy [Hildinger, M et al., "J. Virol" 2001; 75:6199-6203]. Recombinant AAV genomes with AAV2 inverted terminal repeats (ITR) were packaged by triple transfection of 293 cells with the cis plasmid, the adenovirus helper plasmid and a chimeric packaging construct, a fusion of the capsids of the new AAV serotypes with the rep gene of AAV2. The chimeric packaging plasmid was constructed as described previously [Hildinger et al, supra]. The recombinant vectors were purified by the standard CsCb sedimentation method. To determine the yield, TaqMan (Applied Biosystems) analysis was performed using probes and primers targeting the SV40 poly(A) region of the vectors [Gao GP, et al. " Hum Gene Ther." 2000 Oct 10, 11(15):2079-91]. The resulting vectors express the gene in trans under the control of the human thyroid hormone-binding globulin gene promoter (TBG).

B. Animals

LDL receptor-deficient mice with a C57B1/6 background were acquired from Jackson Laboratory (Bar Harbour, ME, USA) and maintained as a breeding colony. The mice were given unlimited access to water and fed a high-fat Western Diet (high % cholesterol) starting three weeks before vector injection. On day -7 as well as day 0, blood was obtained via retroorbital draws and the lipid profile was assessed. The mice were randomly divided into seven groups. The vector was injected via an intraportal injection as described previously ([Chen SJ et al., " Mol Thercpy" 2000; 2(3), 256-261]. Briefly, the mice were anesthetized with ketamine and xylazine. A laparotomy was performed and the portal vein exposed .Using a 30g needle, the appropriate dose of vector, diluted in lOOul PBS, was injected directly into the portal vein. Pressure was applied to the injection site to ensure hemorrhaging. The surrounding skin was closed and draped and the mice were carefully monitored the following day. Weekly draws were conducted starting day 14 after liver-directed gene transfer to measure blood lipids. Two animals in each group were sacrificed at 6 weeks and 12 weeks after vector injection to examine atherosclerotic plaque size as well as receptor expression. The remaining mice were sacrificed at week 20 for plaque measurement and determination of trans gene expression.

C. Serum lipoprotein and liver function assay

Blood samples were obtained from the retroorbital plexus after a 6 h fasting period. Serum was separated from plasma by centrifugation. The amount of plasma lipoproteins and liver transaminases in serum was detected using an automated clinical chemistry analyzer (ACE, Schiapparelli Biosystems, Alpha Wassemann).

D. Detection of transgene expression

LDL receptor expression was evaluated by immunofluorescence staining and Western Blot. For Western Blot, frozen liver tissue was homogenized with lysis buffer (20 mM Tris, pH7.4, 130 mM Nacl, 1% triton X 100, proteinase inhibitor (Complete, EDTA-free, Roche Mannheim, Germany). Protein concentration was determined using "Micro BCA Protein Assay Reagent Kit" (Pierce, Rockford, IL). 40 µg of protein was dissolved in 4-15% Tris-HCl "Ready Gels" (Biorad, Hercules, CA) and transferred to a nitrocellulose membrane (InVitrogen). To generate Anti- hLDL receptor antibodies, a rabbit was injected intravenously with an AdhLDLr prep (lxlO<13>GC). Four weeks later, rabbit serum was obtained and used for Western Blot. A 1:100 dilution of serum was used as a primary antibody followed by an HRP- conjugated anti-rabbit IgG and ECL chemiluminescent detection ("ECL Western Blot Detection Kit", Amersham, Arlington Heights, IL).

E. Immunocytochemistry

To determine the LDL receptor expression in frozen liver sections, immunohistochemistry analyzes were carried out. 10 µm cryostat sections were either fixed in acetone for 5 min or unfixed. Blocking was achieved via a 1 hour incubation period with 10% sheep serum. Sections were then incubated for one hour with the primary antibody at room temperature. A rabbit polyclonal antibody anti-human LDL (Biomedical Technologies Inc., Stoughton, MA) was used diluted according to the manufacturer's instructions. The sections were washed with PBS and incubated with 1:100 diluted fluorescein sheep antirabbit IgG (Sigma, St Louis, MO). Samples were finally examined under a fluorescence microscope Nikon Microphot-FXA. In all cases, each incubation was followed by extensive washing with PBS. Negative controls consisted of preincubation with PBS, omission of the primary antibody, and replacement of the primary antibody with an isotype-matched non-immune control antibody. The three types of controls mentioned above were carried out for each experiment on the same day.

F. Gene transfer efficiency

Liver tissue was obtained after killing the mice at the indicated time points. The tissue was snap-frozen in liquid nitrogen and stored at -80 °C until further processing. DNA was extracted from the liver tissue using a “QIAamp DNA Mini Kit” (QIAGEN Gmbh, Germany) according to the manufacturer's protocol. Genome copy is of AAV vectors in the liver tissue was evaluated using Taqman analysis using probes and primers against the SV40 poly(A) tail as described above.

G. Atherosclerotic plaque measurement

To quantify the presence of atherosclerotic plaque in the mouse aorta, the mice were anesthetized (10% ketamine and xylazine, ip), the chest opened and the arterial system perfused with ice-cold phosphate-buffered saline through the left ventricle. The aorta was then carefully extracted, cut down along the ventral midline from the aortic arch down to the femoral arteries and fixed in formalin. The lipid-rich, atherosclerotic plaques were stained with Sudan IV (Sigma, Germany) and the aorta was mounted flat on a black wax surface. The picture was taken with a Sony DXC-960 MD color video camera. The area of plaque as well as the complete aortic surface was determined using Phase 3 Imaging Systems (Media Cybernetics).

H. Clearance of I<125>LDL

Two animals per experimental group were tested. A bolus of I<125>-labeled LDL (kindly provided by Dan Rader, U Penn) was infused slowly through the needle vein over a period of 30 seconds (1,000,000 counts [1<125>]-LDL diluted in 100 µl sterile PBS/animal. At 3 min, 30 min, 1.5 h, 3 h, and 6 h post-injection, a blood sample was obtained via the retroorbital plexus. Plasma was separated from the whole blood and 10 µl of plasma was counted in the y-counter. To finally, the proportion of catabolic degree was calculated from the obtained lipoprotein clearance data.

I. Evaluation of hepatic lipid accumulation

Oil red staining of frozen liver sections was performed to determine lipid accumulation. The frozen liver sections were briefly rinsed in distilled water followed by a 2 minute incubation period in absolute propylene glycol. Sections were then stained in oil red solution (0.5% in propylene glycol) for 16 hours followed by counterstaining with Mayer's hematoxylin solution for 30 seconds and mounting in warmed glycerin gel solution.

For quantification of liver cholesterol and triglyceride levels, liver sections were homogenized and incubated in chloroform:methanol 2:1 overnight. After addition of 0.05% H2SO4 and centrifugation for 10 minutes, the lower layer of each sample was collected, divided into two aliquots and dried under nitrogen. For the cholesterol measurement, the dried lipids from the first aliquot were dissolved in 1% Triton X-100 in chloroform. Once dissolved, the solution was then dried under nitrogen. After dissolving the lipids in ddH 2 O and incubating for 30 minutes at 37 °C, the total cholesterol concentration was measured using a "Total Cholesterol Kit" (Wako Diagnostics). For the second aliquot, the dried lipids were dissolved in alcoholic KOH and incubated at 60°C for 30 minutes. Then IM MgCl 2 was added, followed by incubation on ice for 10 min and centrifugation at 14,000 rpm for 30 min. The supernatant was finally evaluated for triglycerides (Wako Diagnostics).

All of the vectors pseudotyped in an AAV2/8 or AAV2/7 capsid reduced total cholesterol, LDL, and triglycerides, compared to the control. These test vectors also corrected the phenotype of cholesterolemia in a dose-dependent manner. A reduction of the plaque area for AAV2/8 and AAV2/7 mice was observed in treated mice at the first test (2 months) and the effect was observed to last throughout the experiment (6 months).

Example 10 - Functional factor IX expression and correction of hemophilia

A. Knock-out mice

Functional canine factor IX (FIX) expression was assessed in hemophilia B mice. Vectors with the capsids of AAV1, AAV2, AAV5, AAV7 or AAV8 were constructed to deliver AAV2 5' ITR- liver-specific promoter [LSP] - canine FIX - "woodchuck" hepatitis post-regulatory element (WPRE) - AAV2 3' ITR. The vectors were constructed as described in Wang et al, 2000 "Molecular Therapy" 2: 154-158 using the appropriate capsids.

Knock-out mice were generated as described in Wang et al, 1997 "Proe. Nati. Acad. Sei. USA" 94: 11563-11566. This model closely mimicked the phenotypes of hemophilia B in humans.

Vectors of different serotypes (AAV1, AAV2, AAV5, AAV7 and AAV8) were delivered as a single intraportal injection into the liver of adult hemophilic C57B1/6 mice at a dose of lx10<11>GC/mouse for the five different serotypes and one group received an AAV8 vector at a lower dose, lx10<10>GC/mouse. The control group was injected with lx1011GC of AAV2/8 TBG LacZ3. Each group contained 5-10 male and female mice. The mice were sacrificed every two weeks after vector administration.

1. ELISA

Canine FIX concentration in mouse plasma was determined by an ELISA assay specific for canine factor JX, performed essentially as described by Axelrod et al, 1990 "Proe. Nati. Acad. Sei. USA", 87:5173-5177 with modifications. Sheep anti-canine factor IX (Enzyme Research Laboratories) was used as primary antibody and rabbit anti-canine factor JX (Enzyme Research Laboratories) was used as secondary antibody. Starting two weeks after injection, increased plasma levels of cFJX were determined for all test vectors. The increased levels were maintained at therapeutic levels throughout the length of the trial, ie up to 12 weeks. Therapeutic levels are considered to be 5% of normal levels, i.e. around 250 ng/ml.

The highest levels of expression were observed for the AAV2/8 (at 10<11>) and AAV2/7 constructs that maintained superphysiological levels of cFJX (ten times higher than the normal level). The expression levels for AAV2/8 (IO<11>) were approximately 10-fold higher than the dose observed for AAV2/2 and AAV2/8 (10<10>). The lowest expression levels were observed for AAV2/5 although this was still above the therapeutic range.

2. In vitro activated partial thromboplastin time (aPTT) analysis

Functional factor IV activity in plasma of FDC knock-out mice was determined by an in vitro activated partial thromboplastin time (aPTT) assay. Mouse blood samples were collected from the retroorbital plexus in 1/10 volume citrate buffer. The aPPT assay was performed as described by Wang et al, 1977 in "Proe. Nati. Acad. Sei. USA" 94:11563-11566.

Clotting times by aPTT on plasma samples for all vector-injected mice were within the normal range (approximately 60 seconds), measured two weeks after injection, and maintained clotting times in the normal or shorter than normal range throughout the study period (12 weeks).

The lowest maintained clotting times were observed in the animals receiving AAV2/8 (10<11>) and AAV2/7. After 12 weeks, AAV2/2 also induced clumping times similar to those of AAV2/8 and AAV2/7. However, the lowest clumping times were not observed for AAV2/2 until week 12, while reduced clumping times (in the range of 25 - 40 seconds) were observed for AAV2/8 and AAV2/7 starting at week two.

Immunohistochemistry staining of the liver tissues harvested from some of the treated mice is currently in progress. Approximately 70-80% of the hepatocytes are stained positive for canine FDC in mice injected with AAV2/8.cFDC vector.

B. Hemophilia B dogs

Dogs that have a point mutation in the catalytic domain of the F.DC gene that, based on model studies, appears to make the protein unstable, suffer from hemophilia B (Evans et al, 1989 in "Proe. Nati. Acad. Sei. "USA, 86 :10095-10099). A colony of such dogs has been kept for more than two decades at the University of North Carolina, Chapel Hill. The homeostatic parameters for these dogs are well described and include the absence of plasma F.DC antigen, whole blood clotting times of over 60 minutes while normal dogs are 6-8 minutes, and prolonged activated partial thromboplastin time of 50-80 seconds while normal dogs have 13-28 seconds. These dogs experience recurrent spontaneous haemorrhage. Characteristically, significant bleeding episodes were successfully managed by single intravenous infusion of 10 ml/kg normal canine plasma; occasionally requiring repeated infusions to control bleeding.

Four dogs are injected intraportally with AAV.cFIX according to the schedule below. A lead dog receives a single injection of AAV2/2.cFIX at a dose of 3.7x10<n>genome copies (GC)/kg. A second dog receives a first injection of AAV2/2.cFIX (2.8xl0<n>GC/kg) followed by a second injection of AAV2/7.cFIX (2.3xl0<13>GC/kg) on day 1180. A third dog receives a single injection of AAV2/2.cFIX at a dose of 4.6xl0<12>GC/kg. The fourth dog receives an injection with AAV2/2.cFIX (2.8x10<12>GC/kg) and an injection on day 995 with AAV2/7.cFK (5x1012GC/kg).

The abdomen of hemophiliac dogs is opened aseptically and surgically under general anesthesia and a single infusion of vector is administered into the portal vein. The animals are protected against haemorrhage in the perioperative period by intravenous administration of normal canine plasma. The dogs are sedated, intubated to induce general anesthesia and the abdomen is shaved and prepared. After the abdomen is opened, the spleen is moved to the operating field. The splenic vein is localized and a suture is placed loosely proximal to a small, distal incision in the vein. A needle is quickly inserted into the vein and then the suture is loosened and a 5 F cannula is placed into an intravenous location near the portal vein and advanced to an intravenous location near the portal vein bifurcation. After hemostasis is secured and the catheter balloon inflated, approximately 5.0 ml of vector diluted in PBS is infused into the portal vein over an interval of 5 minutes. The vector infusion is followed by a 5.0 ml saline infusion. The balloon is then deflated, the callula removed and the venous hemostasis ensured. The spleen is then brought back, bleeding vessels cauterized and the surgical wound closed. The animal is extubated and has tolerated the surgical procedure well. The blood samples are analyzed as described [Wang et al, 2000 in "Molecular Therapy" 2: 154-158].

Results showing correction or partial correction are anticipated for AAV2/7.

All publications mentioned in the description must be considered part of it. While the invention is described with reference to particularly preferred embodiments, it should be clear that modifications can be made without going outside the scope of the invention. Such modifications are intended to be within the framework of the requirements.

Claims (13)

1. Adeno-associated virus (AAV), characterized in that it comprises an AAV capsid that has an amino acid sequence AAVrh.10, amino acids 1 to 738 from SEQ ID NO:81 or a sequence that is at least 95% identical thereto and a minigene that has AAV inverted terminal repeats (ITRs) and a heterologous gene operably linked to regulatory sequences that direct its expression in a host cell. 2. Adeno-associated virus (AAV), characterized in that it comprises an AAV capsid comprising at least AAVrh.10 vp3 which has the amino acid sequence 203 to 738 from SEQ ID NO:81 or a sequence which is at least 95% identical thereto and a minigene which has AAV inverted terminal repeats (ITRs) and a heterologous gene operably linked to regulatory sequences that direct its expression in a host cell. 3. Adeno-associated virus (AAV), characterized in that it comprises an AAV capsid comprising at least AAVrh.10 vp2 which has the amino acid sequence 138 to 738 from SEQ ID NO:81 or a sequence which is at least 95% identical thereto and a minigene which has AAV inverted terminal repeats (ITRs) and a heterologous gene operably linked to regulatory sequences that direct its expression in a host cell. 4. AAV according to any of the requirements 1-3, characterized by the fact that the ITRs are from AAV2. 5. Isolated capsid protein, characterized in that it comprises an AAVrh. 10 protein chosen from: Vpl capsid protein, amino acids 1 to 738 of SEQ ID NO:81; and vp2 capsid protein, amino acids 138 to 738 of SEQ ID NO:81; and vp3 capsid protein, amino acids 203 to 738 of SEQ ID NO:81. 6. Isolated or synthetic nucleic acid molecule characterized in that it codes for a protein according to claim 5. 7. Isolated or synthetic nucleic acid molecule encoding a fragment from an adeno-associated virus rh. 10 capsid protein, characterized in that said nucleic acid sequence is selected from the group consisting of: vpl, nucleotides 845 to 3061 of SEQ ID NO:59; vp2, nucleotides 1256 to 3061 of SEQ ID NO:59; and vp3, nucleotides 1454 to 3061 of SEQ ID NO:59. 8. Molecule according to any one of claims 6 or 7, characterized in that said molecule is a plasmid. 9. Molecule according to a health of claims 6 or 7, characterized in that said molecule further comprises a functional AAV rep gene. 10. Method for producing a recombinant adeno-associated virus (AAV), characterized in that it comprises an AAVrh.10 serotype capsid comprising the steps of culturing a host cell containing: a) a molecule according to claims 6 or 7 which codes for an adeno-associated virus capsid; b) a functional rep gene; c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and d) sufficient helper functions to allow packaging of the minigene into the AAV capsid protein. 11. In vitro host cell, characterized in that it is transfected with an adeno-associated virus according to any one of claims 1 to 4 or a molecule according to any one of claims 6 to 9. 12. Composition, characterized in that it comprises AAV according to claim 1 or 4, and a physiologically compatible carrier. 13. Composition characterized in that it comprises a molecule according to any one of claims 6 to 9, and a physiologically compatible carrier.